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Sample records for peptide bond hydrolysis

  1. Effect of organic molecules on hydrolysis of peptide bond: A DFT study

    NASA Astrophysics Data System (ADS)

    Makshakova, Olga; Ermakova, Elena

    2013-03-01

    The activation and inhibition effects of small organic molecules on peptide hydrolysis have been studied using a model compound dialanine and DFT approach. Solvent-assisted and non-assisted concerted mechanisms were analyzed. Several transition states for the systems: alanine dipeptide-water molecule in complexes with alcohol molecules, acetonitrile, dimethylsulfoxide, propionic, lactic and pyruvic acids and water molecules were localized. The formation of hydrogen bonds between dipeptide, reactive water molecule and molecules of solvents influences the activation energy barrier of the peptide bond hydrolytic reaction. Strong effect of organic acids on the activation energy barrier correlates with their electronegativity. Acetonitrile can act as an inhibitor of reaction. Mechanisms of regulation of the activation energy barrier are discussed in the terms of donor-acceptor interactions.

  2. Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering. Combinatorial library determination of optimal sequences.

    PubMed

    Krezel, Artur; Kopera, Edyta; Protas, Anna Maria; Poznański, Jarosław; Wysłouch-Cieszyńska, Aleksandra; Bal, Wojciech

    2010-03-17

    Previously we demonstrated for several examples that peptides having a general internal sequence R(N)-Yaa-Ser/Thr-Xaa-His-Zaa-R(C) (Yaa = Glu or Ala, Xaa = Ala or His, Zaa = Lys, R(N) and R(C) = any N- and C-terminal amino acid sequence) were hydrolyzed specifically at the Yaa-Ser/Thr peptide bond in the presence of Ni(II) ions at alkaline pH (Krezel, A., Mylonas, M., Kopera, E. and Bal, E. Acta Biochim. Polon. 2006, 53, 721-727 and references therein). Hereby we report the synthesis of a combinatorial library of CH(3)CO-Gly-Ala-(Ser/Thr)-Xaa-His-Zaa-Lys-Phe-Leu-NH(2) peptides, where Xaa residues included 17 common alpha-amino acids (except Asp, Glu, and Cys) and Zaa residues included 19 common alpha-amino acids (except Cys). The Ni(II)-dependent hydrolysis at 37 and 45 degrees C of batches of combinatorial peptide mixtures randomized at Zaa was monitored by MALDI-TOF mass spectrometry. The correctness of library-based predictions was confirmed by accurate measurements of hydrolysis rates of seven selected peptides using HPLC. The hydrolysis was strictly limited to the Ala-Ser/Thr bond in all library and individual peptide experiments. The effects of individual residues on hydrolysis rates were quantified and correlated with physical properties of their side chains according to a model of independent contributions of Xaa and Zaa residues. The principal component analysis calculations demonstrated partial molar side chain volume and the free energy of amino acid vaporization for both Xaa and Zaa residues and the amine pK(a) for Zaa residues to be the most significant empirical parameters influencing the hydrolysis rate. Therefore, efficient hydrolysis required bulky and hydrophobic residues at both variable positions Xaa and Zaa, which contributed independently to the hydrolysis rate. This relationship between the peptide sequence and the hydrolysis rate provides a basis for further research, aimed at the elucidation of the reaction mechanism and biotechnological

  3. Molecular mechanism of acid-catalyzed hydrolysis of peptide bonds using a model compound.

    PubMed

    Pan, Bin; Ricci, Margaret S; Trout, Bernhardt L

    2010-04-01

    The stability of peptide bonds is a critical aspect of biological chemistry and therapeutic protein applications. Recent studies found elevated nonenzymatic hydrolysis in the hinge region of antibody molecules, but no mechanism was identified. As a first step in providing a mechanistic interpretation, this computational study examines the rate-determining step of the hydrolytic reaction of a peptide bond under acidic pH by a path sampling technique using a model compound N-MAA. Most previous computational studies did not include explicit water molecules, whose effects are significant in solution chemistry, nor did they provide a dynamic picture for the reaction process in aqueous conditions. Because no single trajectory can be used to describe the reaction dynamics due to fluctuations at finite temperatures, a variant version of the transition path sampling technique, the aimless shooting algorithm, was used to sample dynamic trajectories and to generate an ensemble of transition trajectories according to their statistical weights in the trajectory space. Each trajectory was computed as the time evolution of the molecular system using the Car-Parrinello molecular dynamics technique. The likelihood maximization procedure and its modification were used in extracting dynamically relevant degrees of freedom in the system, and approximations of the reaction coordinate were compared. Its low log-likelihood score and poor p(B) histogram indicate that the C-O distance previously assumed as the reaction coordinate for the rate-determining step is inadequate in describing the dynamics of the reaction. More than one order parameter in a candidate set including millions of geometric quantities was required to produce a convergent reaction coordinate model; its involvement of many degrees of freedom suggests that this hydrolytic reaction step is very complex. In addition to affecting atoms directly involved in bond-making and -breaking processes, the water network also has

  4. Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal

    PubMed Central

    Kopera, Edyta; Belczyk-Ciesielska, Agnieszka; Bal, Wojciech

    2012-01-01

    In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His6. This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques. PMID:22574150

  5. Amide bond hydrolysis in peptides and cyclic peptides catalyzed by a dimeric Zr(IV)-substituted Keggin type polyoxometalate.

    PubMed

    Ly, Hong Giang T; Absillis, Gregory; Parac-Vogt, Tatjana N

    2013-08-14

    Detailed kinetic studies on the hydrolysis of glycylserine (Gly-Ser) and glycylglycine (Gly-Gly) in the presence of the dimeric zirconium(IV)-substituted Keggin type polyoxometalate (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]·7H2O (1) were performed by a combination of (1)H, (13)C and (31)P NMR spectroscopy. The observed rate constants for the hydrolysis of Gly-Ser and Gly-Gly at pD 5.4 and 60 °C were 63.3 × 10(-7) s(-1) and 4.44 × 10(-7) s(-1) respectively, representing a significant acceleration as compared to the uncatalyzed reactions. The pD dependence of the rate constant for both reactions exhibited a bell-shaped profile with the fastest hydrolysis observed in the pD range of 5.5-6.0. Interaction of 1 with Gly-Ser and Gly-Gly via their amine nitrogen and amide oxygen was proven by (13)C NMR spectroscopy. The effective hydrolysis of Gly-Ser in the presence of 1 is most likely a combination of the polarization of the amide oxygen due to its binding to the Zr(IV) ion in 1 and the intramolecular attack of the Ser hydroxyl group on the amide carbonyl carbon. The effect of temperature, inhibitors, and ionic strength on the hydrolysis rate constant was also examined. The solution structure of 1 was investigated by means of (31)P NMR spectroscopy, revealing that its stability is highly dependent on pH, concentration and temperature. A 2.0 mM solution of 1 was found to be fully stable under hydrolytic conditions (pD 5.4 and 60 °C) both in the presence and in the absence of the dipeptides. PMID:23787813

  6. Formation versus Hydrolysis of the Peptide Bond from a Quantum-mechanical Viewpoint: The Role of Mineral Surfaces and Implications for the Origin of Life

    PubMed Central

    Rimola, Albert; Ugliengo, Piero; Sodupe, Mariona

    2009-01-01

    The condensation (polymerization by water elimination) of molecular building blocks to yield the first active biopolymers (e.g. of amino acids to form peptides) during primitive Earth is an intriguing question that nowadays still remains open since these processes are thermodynamically disfavoured in highly dilute water solutions. In the present contribution, formation and hydrolysis of glycine oligopeptides occurring on a cluster model of sanidine feldspar (001) surface have been simulated by quantum mechanical methods. Results indicate that the catalytic interplay between Lewis and Brønsted sites both present at the sanidine surface, in cooperation with the London forces acting between the biomolecules and the inorganic surface, plays a crucial role to: i) favour the condensation of glycine to yield oligopeptides as reaction products; ii) inhibit the hydrolysis of the newly formed oligopeptides. Both facts suggest that mineral surfaces may have helped in catalyzing, stabilizing and protecting from hydration the oligopeptides formed in the prebiotic era. PMID:19399219

  7. Obtaining antimicrobial peptides by controlled peptic hydrolysis of bovine hemoglobin.

    PubMed

    Adje, Estelle Yaba; Balti, Rafik; Kouach, Mostafa; Dhulster, Pascal; Guillochon, Didier; Nedjar-Arroume, Naïma

    2011-08-01

    Under standard conditions, the peptides and specially the active peptides were obtained from either the denatured hemoglobin that all structures are completely modified or either the native hemoglobin where all structures are intact. In these conditions, antibacterial peptides were isolated from a very complex peptidic hydrolysate which contains more than one hundred peptides having various sizes and characteristics, involving a complex purification process. The new hydrolysis conditions were obtained by using 40% methanol, 30% ethanol, 20% propanol or 10% butanol. These conditions, where only the secondary structure of hemoglobin retains intact, were followed in order to enrich the hydrolyzed hemoglobin by active peptides or obtain new antibacterial peptides. In these controlled peptic hydrolysis of hemoglobin, a selective and restrictive hydrolysate contained only 29 peptides was obtained. 26 peptides have an antibacterial activity against Micrococcus luteus, Listeria innocua, and Escherichia coli with MIC from 187.1 to 1 μM. Among these peptides, 13 new antibacterial peptides are obtained only in these new hydrolysis conditions. PMID:21510973

  8. Small peptides hydrolysis in dry-cured meats.

    PubMed

    Mora, Leticia; Gallego, Marta; Escudero, Elizabeth; Reig, Milagro; Aristoy, M-Concepción; Toldrá, Fidel

    2015-11-01

    Large amounts of different peptides are naturally generated in dry-cured meats as a consequence of the intense proteolysis mechanisms which take place during their processing. In fact, meat proteins are extensively hydrolysed by muscle endo-peptidases (mainly calpains and cathepsins) followed by exo-peptidases (mainly, tri- and di-peptidyl peptidases, dipeptidases, aminopeptidases and carboxypeptidases). The result is a large amount of released free amino acids and a pool of numerous peptides with different sequences and lengths, some of them with interesting sequences for bioactivity. This manuscript is presenting the proteomic identification of small peptides resulting from the hydrolysis of four target proteins (glyceraldehyde-3-phosphate dehydrogenase, beta-enolase, myozenin-1 and troponin T) and discusses the enzymatic routes for their generation during the dry-curing process. The results indicate that the hydrolysis of peptides follows similar exo-peptidase mechanisms. In the case of dry-fermented sausages, most of the observed hydrolysis is the result of the combined action of muscle and microbial exo-peptidases except for the hydrolysis of di- and tri-peptides, mostly due to microbial di- and tri-peptidases, and the release of amino acids at the C-terminal that appears to be mostly due to muscle carboxypeptidases. PMID:25944374

  9. Catalytic peptide hydrolysis by mineral surface: Implications for prebiotic chemistry

    NASA Astrophysics Data System (ADS)

    Marshall-Bowman, Karina; Ohara, Shohei; Sverjensky, Dimitri A.; Hazen, Robert M.; Cleaves, H. James

    2010-10-01

    The abiotic polymerization of amino acids may have been important for the origin of life, as peptides may have been components of the first self-replicating systems. Though amino acid concentrations in the primitive oceans may have been too dilute for significant oligomerization to occur, mineral surface adsorption may have provided a concentration mechanism. As unactivated amino acid polymerization is thermodynamically unfavorable and kinetically slow in aqueous solution, we studied mainly the reverse reaction of polymer degradation to measure the impact of mineral surface catalysis on peptide bonds. Aqueous glycine (G), diglycine (GG), diketopiperazine (DKP), and triglycine (GGG) were reacted with minerals (calcite, hematite, montmorillonite, pyrite, rutile, or amorphous silica) in the presence of 0.05 M, pH 8.1, KHCO 3 buffer and 0.1 M NaCl as background electrolyte in a thermostatted oven at 25, 50 or 70 °C. Below 70 °C, reaction kinetics were too sluggish to detect catalytic activity over amenable laboratory time-scales. Minerals were not found to have measurable effects on the degradation or elongation of G, GG or DKP at 70 °C in solution. At 70 °C pyrite was the most catalytic mineral with detectible effects on the degradation of GGG, although several others also displayed catalytic behavior. GGG degraded ˜1.5-4 times faster in the presence of pyrite than in control reactions, depending on the ratio of solution concentration to mineral surface area. The rate of pyrite catalysis of GGG hydrolysis was found to be saturable, suggesting the presence of discrete catalytic sites on the mineral surface. The mineral-catalyzed degradation of GGG appears to occur via a GGG → DKP + G mechanism, rather than via GGG → GG + G, as in solution-phase reactions. These results are compatible with many previous findings and suggest that minerals may have assisted in peptide synthesis in certain geological settings, specifically by speeding the approach to equilibrium

  10. Catalytic RNA and synthesis of the peptide bond

    NASA Technical Reports Server (NTRS)

    Usher, D. A.; Kozlowski, M.; Zou, X.

    1991-01-01

    We are studying whether the L-19 IVS ribozyme from Tetrahymena thermophila can catalyze the formation of the peptide bond when it is supplied with synthetic aminoacyl oligonucleotides. If this reaction works, it could give us some insight into the mechanism of peptide bond formation and the origin of coded protein synthesis. Two short oligoribonucleotides, CCCCC and a protected form of CCCCU were prepared; the former was made by the controlled hydrolysis of Poly(C), and the later by multistep chemical synthesis from the protected monomers. The homopentamer was then aminocylated using C-14 labelled Boc-protected glycine imidazolide. This aminoacylated oligo-nucleotide has now been shown to enter the active site of the L-19 IVS, and aminoacyl transfer, and peptide bond formation reactions are being sought. Our synthesis of CCCCU made us aware of the inadequacy of many of the 2'- hydroxyl protecting groups that are in use today and we therefore designed a new 2'- protecting group that is presently being tested.

  11. Benchmarking of DFT Functionals for the Hydrolysis of Phosphodiester Bonds.

    PubMed

    Ribeiro, António J M; Ramos, Maria J; Fernandes, Pedro A

    2010-08-10

    Phosphodiester bonds are an important chemical component of biological systems, and their hydrolysis and formation reactions are involved in major steps throughout metabolic pathways of all organisms. In this work, we applied dimethylphosphate as a model for this kind of bonds and calculated the potential energy surface for its hydrolysis at the approximated CCSD(T)/CBS//B3LYP/6-311++G(2d,2p) level. By varying the nucleophile (water or hydroxide) and the medium (vacuum or aqueous implicit solvent) we obtained and described four reaction paths. These structures were then used in a DFT functional benchmarking in which we tested a total of 52 functionals. Furthermore, the performances of HF, MP2, MP3, MP4, and CCSD were also evaluated. This benchmarking showed that MPWB1K, MPW1B95, and PBE1PBE are the more accurate functionals to calculate the energies of dimethylphosphate hydrolysis as far as activation and reaction energies are concerned. If considering only the activation energies, MPWB1K, MPW1B95, and B1B95 give the lowest errors when comparing to CCSD(T). A basis set benchmarking on the same system shows that 6-311+G(2d,2p) is the best basis set concerning the relationship between computational time and accuracy. We believe that our results will be of great help to further studies on related phosphodiester systems. This includes not only pure chemical problems but also biochemical studies in which DNA, RNA, and phospholipids are required to be depicted at a quantum level. PMID:26613486

  12. Structural analysis of peptide fragments following the hydrolysis of bovine serum albumin by trypsin and chymotrypsin.

    PubMed

    Özyiğit, İbrahim Ethem; Akten, E Demet; Pekcan, Önder

    2016-05-01

    Peptide bond hydrolysis of bovine serum albumin (BSA) by chymotrypsin and trypsin was investigated by employing time-resolved fluorescence spectroscopy. As a fluorescent cross-linking reagent, N-(1-pyrenyl) maleimide (PM) was attached to BSA, through all free amine groups of arginine, lysine, and/or single free thiol (Cys34). Time-resolved fluorescence spectroscopy was used to monitor fluorescence decays analyzed by exponential series method to obtain the changes in lifetime distributions. After the exposure of synthesized protein substrate PM-BSA to chymotrypsin and trypsin, it is observed that each protease produced a distinct change in the lifetime distribution profile, which was attributed to distinct chemical environments created by short peptide fragments in each hydrolysate. The persistence of excimer emission at longer lifetime regions for chymotrypsin, as opposed to trypsin, suggested the presence of small-scale hydrophobic clusters that might prevent some excimers from being completely quenched. It is most likely that the formation of these clusters is due to hydrophobic end groups of peptide fragments in chymotrypsin hydrolysate. A similar hydrophobic shield was not suggested for trypsin hydrolysis, as the end groups of peptide fragments would be either arginine or lysine. Overall, in case the target protein's 3D structure is known, the structural analysis of possible excimer formation presented here can be used as a tool to explain the differences in activity between two proteases, i.e. the peak's intensity and location in the profile. Furthermore, this structural evaluation might be helpful in obtaining the optimum experimental conditions in order to generate the highest amount of PM-BSA complexes. PMID:26169062

  13. Amino acid side chain induced selectivity in the hydrolysis of peptides catalyzed by a Zr(IV)-substituted Wells-Dawson type polyoxometalate.

    PubMed

    Vanhaecht, Stef; Absillis, Gregory; Parac-Vogt, Tatjana N

    2013-11-21

    In this paper the reactivity of K15H[Zr(α2-P2W17O61)2]·25H2O (1), a Zr(IV)-substituted Wells-Dawson polyoxometalate, is examined towards a series of Gly-Aa, Aa-Gly or Aa-Ser dipeptides, in which the nature and the size of the Aa amino acid side chain were varied. The rate of peptide bond hydrolysis, determined by (1)H NMR experiments, in Gly-Aa dipeptides is strongly dependent on the molecular volume and the chemical structure of the Aa side chain. When the volume of the aliphatic side chain of the Aa residue in Gly-Aa increased, a clear decrease in the hydrolysis rate was observed. Replacing one α-H in the C-terminal Gly residue of Gly-Gly by a methyl group (Gly-Ala) resulted in a 6-fold reactivity decrease, pointing towards the importance of steric factors for efficient peptide bond hydrolysis. The rate constants for peptide bond hydrolysis in Gly-Aa dipeptides at pD 5.0 and 60 °C ranged from 208.0 ± 15.6 × 10(-6) min(-1) for Gly-Ser to 5.0 ± 1.0 × 10(-6) min(-1) for Gly-Glu, reflecting the influence of the different nature of the amino acid side chains on the hydrolysis rate. Faster hydrolysis was observed for peptides containing Ser and Thr since the hydroxyl group in their side chain is able to facilitate amide bond hydrolysis by promoting an N→O acyl rearrangement. Peptides containing positively charged side chains at pD 5.0 show enhanced hydrolysis rates as a result of the secondary electrostatic interactions with the negatively charged surface of the polyoxometalate, which stabilize the peptide-polyoxometalate complex. A slow hydrolysis rate was observed for Gly-Glu, because of the preferential coordination of the carboxylate group in the side chain of Glu to Zr(IV), which prevents coordination of the peptide carbonyl group and its activation towards hydrolysis. PMID:24018583

  14. Enzymatic Hydrolysis of Peanut Flour Produces Bioactive Peptides with Reduced Allergenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peanut allergy is one of the most severe food allergies due to its life-threatening nature and persistency. Current immunotherapy methods, though effective, are often accompanied by allergic side-effects. Enzymatic hydrolysis of peanut flour has the potential to produce bioactive peptides with impro...

  15. Influence of heat pre-treatment on BSA tryptic hydrolysis and peptide release.

    PubMed

    Arrutia, Fátima; Puente, Ángela; Riera, Francisco A; Menéndez, Carlos; González, Ulises A

    2016-07-01

    In contrast with other food proteins, such as β-lactoglobulin or caseins, intensely studied for bioactive peptide production, relatively little attention has been paid to serum albumin, the main blood protein, even though blood disposal is a severe problem for meat processors. In this study, serum albumin was hydrolysed with trypsin after several heat treatments and using different enzyme concentrations. The degree of hydrolysis reached and the peptide sequences released over time were evaluated. Large differences in enzyme-to-substrate ratios (1:50, 1:100 and 1:200) led to similar degree of hydrolysis values (31.92±1.43%, 31.08±3.09% and 26.21±0.71%), and did not alter the number of peptides released. However, thermal treatment enhanced significantly (p<0.05) both the degree of hydrolysis (up to 50.41±1.90%) and the number and amount of the majority of peptides obtained, all with potential bioactivity (28 peptides in the native hydrolysate, 39 in the thermally treated). PMID:26920264

  16. Peptide sequencing by using a combination of partial acid hydrolysis and fast-atom-bombardment mass spectrometry.

    PubMed Central

    De Angelis, F; Botta, M; Ceccarelli, S; Nicoletti, R

    1986-01-01

    To overcome the limit of the intensity of ions carrying sequence information in structural determinations of peptides by fast-atom-bombardment m.s., we have developed a method that consists in taking spectra of the peptide acid hydrolysates at different hydrolysis times. Peaks correspond to the oligomers arising from the peptide partial hydrolysis. The sequence can then be identified from the structurally overlapping fragments. PMID:2428356

  17. Evolutionary Importance of the Intramolecular Pathways of Hydrolysis of Phosphate Ester Mixed Anhydrides with Amino Acids and Peptides

    NASA Astrophysics Data System (ADS)

    Liu, Ziwei; Beaufils, Damien; Rossi, Jean-Christophe; Pascal, Robert

    2014-12-01

    Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO2-promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO2-promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide-phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.

  18. Enzyme-substrate interactions in the hydrolysis of peptides by cathepsins B and H from rat liver.

    PubMed Central

    Brömme, D; Bescherer, K; Kirschke, H; Fittkau, S

    1987-01-01

    The number of possible subsites of the rat liver cysteine proteinases cathepsin B and cathepsin H was determined in the N-terminal direction from the scissile bond. An elongation of the substrate peptide chain of up to four amino acid residues enhances the hydrolysis rate of both cathepsins. The greatest increase in activity was observed by elongation to the dipeptide substrate for cathepsin B and to the tetrapeptide substrate for cathepsin H. Both proteinases discriminate proline from their subsites S1 and S2, but accept it well in S3. A quantitative distinction between the endopeptidase and the peptidyl dipeptidase activity of cathepsin B was feasible by using two model peptides: (Formula: see text) (Z = benzyloxycarbonyl; X = NH2 or OH; the arrow shows the cleavage site). Whereas the peptide acid, representing the peptidyl dipeptidase substrate, was hydrolysed by cathepsin B twice as fast as the peptide amide as an endopeptidase substrate, cathepsin H clearly had a preference for the amide substrate. PMID:3663163

  19. Controlled Enzymatic Hydrolysis: A New Strategy for the Discovery of Antimicrobial Peptides.

    PubMed

    Adje, Estelle Yaba; Balti, Rafik; Lecouturier, Didier; Kouach, Mostafa; Dhulster, Pascal; Guillochon, Didier; Nedjar-Arroume, Naïma

    2013-09-01

    The use of antimicrobial peptides (AMPs) is an alternative to traditional antibiotics. AMPs are obtained using different methods such as bacterial synthesis, chemical synthesis and controlled enzymatic hydrolysis. The later is an interesting approach that deserves our attention because of the yields gathered and peptides engineered. Usually, activities of AMPs obtained in such a way are tightly dependent on the hydrolysis mechanism used. This paper deals with the hydrolysis of hemoglobin mechanism as a potential source of AMPs. Production of AMPs from hemoglobin using enzymatic controlled system is linked to hemoglobin structure. Further, we show that bovine hemoglobin, which is sensitive to peptic hydrolysis, results upon enzymatic digestion as a great source of AMPs. The hemoglobin in native and denatured states was hydrolyzed by "one-by-one" and "zipper" mechanisms, respectively. Nevertheless, a new mechanism named "semi-zipper" mechanism is obtained when protein is in molten globule structural state, constituting an original strategy for AMPs production. Seventy seven percentage of the peptides obtained by this new strategy showed antibacterial activity against nine strains. PMID:26782986

  20. Disulfide bond assignments by mass spectrometry of native natural peptides: cysteine pairing in disulfide bonded conotoxins.

    PubMed

    Gupta, Kallol; Kumar, Mukesh; Balaram, Padmanabhan

    2010-10-01

    The critical, and often most difficult, step in structure elucidation of diverse classes of natural peptides is the determination of correct disulfide pairing between multiple cysteine residues. Here, we present a direct mass spectrometric analytical methodology for the determination of disulfide pairing. Protonated peptides, having multiple disulfide bonds, fragmented under collision induced dissociation (CID) conditions and preferentially cleave along the peptide backbone, with occasional disulfide fragmentation either by C(β)-S bond cleavage through H(α) abstraction to yield dehydroalanine and cysteinepersulfide, or by S-S bond cleavage through H(β) abstraction to yield the thioaldehyde and cysteine. Further fragmentation of the initial set of product ions (MS(n)) yields third and fourth generation fragment ions, permitting a distinction between the various possible disulfide bonded structures. This approach is illustrated by establishing cysteine pairing patterns in five conotoxins containing two disulfide bonds. The methodology is extended to the Conus araneosus peptides Ar1446 and Ar1430, two 14 residue sequences containing 3 disulfide bonds. A distinction between 15 possible disulfide pairing schemes becomes possible using direct mass spectral fragmentation of the native peptides together with fragmentation of enzymatically nicked peptides. PMID:20843009

  1. Peptide Bond Formation in Water Mediated by Carbon Disulfide.

    PubMed

    Leman, Luke J; Huang, Zheng-Zheng; Ghadiri, M Reza

    2015-09-01

    Demonstrating plausible nonenzymatic polymerization mechanisms for prebiotic monomers represents a fundamental goal in prebiotic chemistry. While a great deal is now known about the potentially prebiotic synthesis of amino acids, our understanding of abiogenic polymerization processes to form polypeptides is less well developed. Here, we show that carbon disulfide (CS2), a component of volcanic emission and sulfide mineral weathering, and a widely used synthetic reagent and solvent, promotes peptide bond formation in modest yields (up to ∼20%) from α-amino acids under mild aqueous conditions. Exposure of a variety of α-amino acids to CS2 initially yields aminoacyl dithiocarbamates, which in turn generate reactive 2-thiono-5-oxazolidone intermediates, the thio analogues of N-carboxyanhydrides. Along with peptides, thiourea and thiohydantoin species are produced. Amino acid stereochemistry was preserved in the formation of peptides. Our findings reveal that CS2 could contribute to peptide bond formation, and possibly other condensation reactions, in abiogenic settings. PMID:26308392

  2. Peptide Bond Isomerization in High-Temperature Simulations.

    PubMed

    Neale, Chris; Pomès, Régis; García, Angel E

    2016-04-12

    Force fields for molecular simulation are generally optimized to model macromolecules such as proteins at ambient temperature and pressure. Nevertheless, elevated temperatures are frequently used to enhance conformational sampling, either during system setup or as a component of an advanced sampling technique such as temperature replica exchange. Because macromolecular force fields are now put upon to simulate temperatures and time scales that greatly exceed their original design specifications, it is appropriate to re-evaluate whether these force fields are up to the task. Here, we quantify the rates of peptide bond isomerization in high-temperature simulations of three octameric peptides and a small fast-folding protein. We show that peptide octamers with and without proline residues undergo cis/trans isomerization every 1-5 ns at 800 K with three classical atomistic force fields (AMBER99SB-ILDN, CHARMM22/CMAP, and OPLS-AA/L). On the low microsecond time scale, these force fields permit isomerization of nonprolyl peptide bonds at temperatures ≥500 K, and the CHARMM22/CMAP force field permits isomerization of prolyl peptide bonds ≥400 K. Moreover, the OPLS-AA/L force field allows chiral inversion about the Cα atom at 800 K. Finally, we show that temperature replica exchange permits cis peptide bonds developed at 540 K to subsequently migrate back to the 300 K ensemble, where cis peptide bonds are present in 2 ± 1% of the population of Trp-cage TC5b, including up to 4% of its folded state. Further work is required to assess the accuracy of cis/trans isomerization in the current generation of protein force fields. PMID:26866899

  3. Bioactive properties of peptides obtained from Argentinian defatted soy flour protein by Corolase PP hydrolysis.

    PubMed

    Coscueta, Ezequiel R; Amorim, Maria M; Voss, Glenise B; Nerli, Bibiana B; Picó, Guillermo A; Pintado, Manuela E

    2016-05-01

    Enzymatic hydrolysis of soybean meal protein isolate (SPI) obtained under two temperature conditions with Corolase PP was studied, assessing the impact of hydrolysis on potential antioxidant and antihypertensive activities. The protein was isolated from soybean meal under controlled conditions of time and temperature (70 °C, 1h; 90 °C, 30 min). Degree of hydrolysis assessed the progress of hydrolysis at different sampling times. For hydrolysates the antioxidant and angiotensin-converting-enzyme (ACE) inhibitory activities were measured. As observed, the DH was increasing until reaching 20% at 10h with disappearance of globular proteins and generation of low molecular weight peptides (less than 3kDa). A significant increase in antioxidant and ACE inhibitory capacities was observed. Five main peptides were identified, which may explain through their sequences the bioactive properties analyzed. Through this study was possible to obtain for the first time with Corolase PP soy hydrolysates with potential antioxidant and ACE inhibitory activities, which can be used to obtain new added value functional ingredients from soy meal. PMID:26769502

  4. Use of an electrodialytic reactor for the simultaneous β-lactoglobulin enzymatic hydrolysis and fractionation of generated bioactive peptides.

    PubMed

    Doyen, Alain; Husson, Eric; Bazinet, Laurent

    2013-02-15

    The enzymatic hydrolysis of β-lactoglobulin and the fractionation of peptides were performed in one step in an electrodialysis cell with ultrafiltration membranes stacked. After 240 min of treatment, 15 anionic and 4 cationic peptides were detected in the anionic and cationic peptide recovery compartments. Amongst these 15 anionic peptides, 2 hypocholesterolemic, 3 antihypertensive and 1 antibacterial peptides were recovered and concentrated with migration rates ranging from 5.5% and 81.7%. Amongst the 4 cationic peptides, the peptide sequence ALPMHIR, identified as lactokinin and known to exert an important antihypertensive effect, was recovered with an estimated 66% migration rate. To our knowledge, it was the first attempt to perform hydrolysis under an electric field and to simultaneously separate anionic and cationic peptides produced. PMID:23194514

  5. Evolutionary Importance of the Intramolecular Pathways of Hydrolysis of Phosphate Ester Mixed Anhydrides with Amino Acids and Peptides

    PubMed Central

    Liu, Ziwei; Beaufils, Damien; Rossi, Jean-Christophe; Pascal, Robert

    2014-01-01

    Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO2-promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO2-promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide–phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation. PMID:25501391

  6. Interplay between Peptide Bond Geometrical Parameters in Nonglobular Structural Contexts

    PubMed Central

    Esposito, Luciana; De Simone, Alfonso; Vitagliano, Luigi

    2013-01-01

    Several investigations performed in the last two decades have unveiled that geometrical parameters of protein backbone show a remarkable variability. Although these studies have provided interesting insights into one of the basic aspects of protein structure, they have been conducted on globular and water-soluble proteins. We report here a detailed analysis of backbone geometrical parameters in nonglobular proteins/peptides. We considered membrane proteins and two distinct fibrous systems (amyloid-forming and collagen-like peptides). Present data show that in these systems the local conformation plays a major role in dictating the amplitude of the bond angle N-Cα-C and the propensity of the peptide bond to adopt planar/nonplanar states. Since the trends detected here are in line with the concept of the mutual influence of local geometry and conformation previously established for globular and water-soluble proteins, our analysis demonstrates that the interplay of backbone geometrical parameters is an intrinsic and general property of protein/peptide structures that is preserved also in nonglobular contexts. For amyloid-forming peptides significant distortions of the N-Cα-C bond angle, indicative of sterical hidden strain, may occur in correspondence with side chain interdigitation. The correlation between the dihedral angles Δω/ψ in collagen-like models may have interesting implications for triple helix stability. PMID:24455689

  7. cis-Peptide Bonds: A Key for Intestinal Permeability of Peptides? .

    PubMed

    Marelli, Udaya Kiran; Ovadia, Oded; Frank, Andreas Oliver; Chatterjee, Jayanta; Gilon, Chaim; Hoffman, Amnon; Kessler, Horst

    2015-10-19

    Recent structural studies on libraries of cyclic hexapeptides led to the identification of common backbone conformations that may be instrumental to the oral availability of peptides. Furthermore, the observation of differential Caco-2 permeabilities of enantiomeric pairs of some of these peptides strongly supports the concept of conformational specificity driven uptake and also suggests a pivotal role of carrier-mediated pathways for peptide transport, especially for scaffolds of polar nature. This work presents investigations on the Caco-2 and PAMPA permeability profiles of 13 selected N-methylated cyclic pentaalanine peptides derived from the basic cyclo(-D-Ala-Ala4 -) template. These molecules generally showed moderate to low transport in intestinal epithelia with a few of them exhibiting a Caco-2 permeability equal to or slightly higher than that of mannitol, a marker for paracellular permeability. We identified that the majority of the permeable cyclic penta- and hexapeptides possess an N-methylated cis-peptide bond, a structural feature that is also present in the orally available peptides cyclosporine A and the tri-N-methylated analogue of the Veber-Hirschmann peptide. Based on these observations it appears that the presence of N-methylated cis-peptide bonds at certain locations may promote the intestinal permeability of peptides through a suitable conformational preorganization. PMID:26337831

  8. Site-selective chemical cleavage of peptide bonds.

    PubMed

    Elashal, Hader E; Raj, Monika

    2016-05-01

    Site-selective cleavage of extremely unreactive peptide bonds is a very important chemical modification that provides invaluable information regarding protein sequence, and it acts as a modulator of protein structure and function for therapeutic applications. For controlled and selective cleavage, a daunting task, chemical reagents must selectively recognize or bind to one or more amino acid residues in the peptide chain and selectively cleave a peptide bond. Building on this principle, we have developed an approach that utilizes a chemical reagent to selectively modify the serine residue in a peptide chain and leads to the cleavage of a peptide backbone at the N-terminus of the serine residue. After cleavage, modified residues can be converted back to the original fragments. This method exhibits broad substrate scope and selectively cleaves various bioactive peptides with post-translational modifications (e.g. N-acetylation and -methylation) and mutations (d- and β-amino acids), which are a known cause of age related diseases. PMID:27087443

  9. Ribosomal crystallography: peptide bond formation and its inhibition.

    PubMed

    Bashan, Anat; Zarivach, Raz; Schluenzen, Frank; Agmon, Ilana; Harms, Joerg; Auerbach, Tamar; Baram, David; Berisio, Rita; Bartels, Heike; Hansen, Harly A S; Fucini, Paola; Wilson, Daniel; Peretz, Moshe; Kessler, Maggie; Yonath, Ada

    2003-09-01

    Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis. The large subunit creates the peptide bond and provides the path for emerging proteins. The small has key roles in initiating the process and controlling its fidelity. Crystallographic studies on complexes of the small and the large eubacterial ribosomal subunits with substrate analogs, antibiotics, and inhibitors confirmed that the ribosomal RNA governs most of its activities, and indicated that the main catalytic contribution of the ribosome is the precise positioning and alignment of its substrates, the tRNA molecules. A symmetry-related region of a significant size, containing about two hundred nucleotides, was revealed in all known structures of the large ribosomal subunit, despite the asymmetric nature of the ribosome. The symmetry rotation axis, identified in the middle of the peptide-bond formation site, coincides with the bond connecting the tRNA double-helical features with its single-stranded 3' end, which is the moiety carrying the amino acids. This thus implies sovereign movements of tRNA features and suggests that tRNA translocation involves a rotatory motion within the ribosomal active site. This motion is guided and anchored by ribosomal nucleotides belonging to the active site walls, and results in geometry suitable for peptide-bond formation with no significant rearrangements. The sole geometrical requirement for this proposed mechanism is that the initial P-site tRNA adopts the flipped orientation. The rotatory motion is the major component of unified machinery for peptide-bond formation, translocation, and nascent protein progression, since its spiral nature ensures the entrance of the nascent peptide into the ribosomal exit tunnel. This tunnel, assumed to be a passive path for the

  10. Vibrational characterization of the peptide bond

    NASA Astrophysics Data System (ADS)

    Herrebout, W.; Clou, K.; Desseyn, H. O.; Blaton, N.

    2003-01-01

    This article describes the complete vibrational analysis of N, N'-dimethyloxamide, CH 3HNCOCONHCH 3, on basis of the infrared and Raman spectra of four isotopes (H, D, CH 3, CD 3). Force field calculations on the monomers and multimers ( n=5) combined with solid state spectra in the -196 to +100 °C temperature range have been used to obtain a better understanding of the influence of hydrogen bonding on the typical amide fundamentals. The cooperative effect in de series monomer→multimers→solid state at decreasing temperatures has been demonstrated. Nine typical so-called 'amide bands' have been further characterized and special attention has been given to the Amide IV mode. The influence of the CH and CD vibrations on the amide fundamentals, has been studied by comparison with the calculated and experimental fundamentals and P.E.D. values of the CH 3 and CD 3 isotopes. The most important amide bands have further been assigned in X-CONHCH 3 molecules where X=methyl, amide, thioamide, ester, salt, cyanide and acid functional groups.

  11. Vibrational characterization of the peptide bond.

    PubMed

    Herrebout, W; Clou, K; Desseyn, H O; Blaton, N

    2003-01-01

    This article describes the complete vibrational analysis of N,N'-dimethyloxamide, CH3HNCOCONHCH3, on basis of the infrared and Raman spectra of four isotopes (H, D, CH3, CD3). Force field calculations on the monomers and multimers (n = 5) combined with solid state spectra in the -196 to +100 degrees C temperature range have been used to obtain a better understanding of the influence of hydrogen bonding on the typical amide fundamentals. The cooperative effect in de series monomer --> multimers --> solid state at decreasing temperatures has been demonstrated. Nine typical so-called 'amide bands' have been further characterized and special attention has been given to the Amide IV mode. The influence of the CH and CD vibrations on the amide fundamentals, has been studied by comparison with the calculated and experimental fundamentals and P.E.D. values of the CH3 and CD3 isotopes. The most important amide bands have further been assigned in X-CONHCH3 molecules where X = methyl, amide, thioamide, ester, salt, cyanide and acid functional groups. PMID:12509146

  12. N-Acetylglycine Cation Tautomerization Enabled by the Peptide Bond.

    PubMed

    Kocisek, Jaroslav; Piekarski, Dariusz Grzegorz; Delaunay, Rudy; Huber, Bernd A; Adoui, Lamri; Martín, Fernando; Alcamí, Manuel; Rousseau, Patrick; Domaracka, Alicja; Kopyra, Janina; Díaz-Tendero, Sergio

    2015-09-17

    We present a combined experimental and theoretical study of the ionization of N-acetylglycine molecules by 48 keV O(6+) ions. We focus on the single ionization channel of this interaction. In addition to the prompt fragmentation of the N-acetylglycine cation, we also observe the formation of metastable parent ions with lifetimes in the microsecond range. On the basis of density functional theory calculations, we assign these metastable ions to the diol tautomer of N-acetylglycine. In comparison with the simple amino acids, the tautomerization rate is higher because of the presence of the peptide bond. The study of a simple biologically relevant molecule containing a peptide bond allows us to demonstrate how increasing the complexity of the structure influences the behavior of the ionized molecule. PMID:26243533

  13. Amide and Peptide Bond Formation in Water at Room Temperature.

    PubMed

    Gabriel, Christopher M; Keener, Megan; Gallou, Fabrice; Lipshutz, Bruce H

    2015-08-21

    A general and environmentally responsible method for the formation of amide/peptide bonds in an aqueous micellar medium is described. Use of uronium salt (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU) as a coupling reagent, 2,6-lutidine, and TPGS-750-M represents mild conditions associated with these valuable types of couplings. The aqueous reaction medium is recyclable leading to low E Factors. PMID:26251952

  14. Valorization of cruor slaughterhouse by-product by enzymatic hydrolysis for the production of antibacterial peptides: focus on α 1-32 family peptides mechanism and kinetics modeling.

    PubMed

    Hedhili, K; Dimitrov, K; Vauchel, P; Sila, A; Chataigné, G; Dhulster, P; Nedjar, N

    2015-10-01

    Bovine hemoglobin is the major component of the cruor (slaughterhouse by-product) and can be considered as an important source of active peptides that could be obtained by pepsic hydrolysis. The kinetics of appearance and disappearance of several antibacterial peptides from α 1-32 family during hydrolysis of synthesized α 1-32 peptide, of purified bovine hemoglobin and of cruor was studied, and reaction scheme for the hydrolysis of α 1-32 family peptides from these three sources was determined. On this basis, a mathematical model was proposed to predict the concentration of each peptide of interest of this family depending on hydrolysis time, and also on temperature (in the range 15-37 °C), pH (in the range 3.5-5.5) and enzyme to substrate ratio (in the range 1/50-1/200 for the synthesized peptide and 1/5-1/20 for purified bovine hemoglobin and cruor). Apparent rate constants of reactions were determined by applying the model on a set of experimental data and it was shown that they depended on the temperature according to Arrhenius's law, that their dependence on the pH was linear, and that enzyme to substrate ratio influence was limited (in the studied range). PMID:26099509

  15. Peptide-Like Molecules (PLMs): A Journey from Peptide Bond Isosteres to Gramicidin S Mimetics and Mitochondrial Targeting Agents

    PubMed Central

    Wipf, Peter; Xiao, Jingbo; Stephenson, Corey R. J.

    2010-01-01

    Peptides are natural ligands and substrates for receptors and enzymes and exhibit broad physiological effects. However, their use as therapeutic agents often suffers from poor bioavailability and insufficient membrane permeability. The success of peptide mimicry hinges on the ability of bioisosteres, in particular peptide bond replacements, to adopt suitable secondary structures relevant to peptide strands and position functional groups in equivalent space. This perspective highlights past and ongoing studies in our group that involve new methods development as well as specific synthetic library preparations and applications in chemical biology, with the goal to enhance the use of alkene and cyclopropane peptide bond isosteres. PMID:20725595

  16. Partial Molecular Characterization of Arctium minus Aspartylendopeptidase and Preparation of Bioactive Peptides by Whey Protein Hydrolysis.

    PubMed

    Cimino, Cecilia V; Colombo, María Laura; Liggieri, Constanza; Bruno, Mariela; Vairo-Cavalli, Sandra

    2015-08-01

    In this article, we report the cloning of an aspartic protease (AP) from flowers of Arctium minus (Hill) Bernh. (Asteraceae) along with the use of depigmented aqueous flower extracts, as a source of APs, for the hydrolysis of whey proteins. The isolated cDNA encoded a protein product with 509 amino acids called arctiumisin, with the characteristic primary structure organization of typical plant APs. Bovine whey protein hydrolysates, obtained employing the enzyme extracts of A. minus flowers, displayed inhibitory angiotensin-converting enzyme (ACE) and antioxidant activities. Hydrolysates after 3 and 5 h of reaction (degree of hydrolysis 2.4 and 5.6, respectively) and the associated peptide fraction with molecular weight below 3 kDa were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization/time of flight mass spectrometry, and reverse phase-high-performance liquid chromatography. The results obtained in this study demonstrate the viability of using proteases from A. minus to increase the antioxidant and inhibitory ACE capacity of whey proteins. PMID:25575270

  17. Influence of denaturation and aggregation of β-lactoglobulin on its tryptic hydrolysis and the release of functional peptides.

    PubMed

    Leeb, Elena; Götz, Alexander; Letzel, Thomas; Cheison, Seronei Chelulei; Kulozik, Ulrich

    2015-11-15

    Whereas previous studies showed that thermal pre-treatment of whey proteins promote their enzymatic hydrolysis, to date no correlation between the conformation of denatured protein and the release of individual peptides has been considered. Hence, in this study total denaturation of β-lactoglobulin was performed at defined pH-values to enable the generation of different denatured particles. The denatured proteins were used as substrate for tryptic hydrolysis and the hydrolysis progress was characterised by the degree of hydrolysis (DH) and the release of functional peptides, detected using LC-ESI-TOF/MS. Denaturation and subsequent aggregation of β-lactoglobulin, induced by thermal treatment at pH 5.1, altered the DH slightly, whereas the release of investigated peptides was significantly decreased. Contrary, denaturation at pH 6.8 and 8.0 led to formation of non-native monomers and reduced the DH to 75%, but showed promoting as well as reducing effects on the release of peptides, depending on their location within the protein. PMID:25977062

  18. Performance of coupled enzymatic hydrolysis and membrane separation bioreactor for antihypertensive peptides production from Porphyra yezoensis protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To explore more efficient production methods of antihypertensive peptides from Porphyra yezoensis protein, three methods of coupling of enzymatic hydrolysis and membrane separation (CEH-MS) were studied and compared with the traditional EH and offline MS method. The results showed that the conversio...

  19. Modulation of reactivity in the cavity of liposomes promotes the formation of peptide bonds.

    PubMed

    Grochmal, Anna; Prout, Luba; Makin-Taylor, Robert; Prohens, Rafel; Tomas, Salvador

    2015-09-30

    In living cells, reactions take place in membrane-bound compartments, often in response to changes in the environment. Learning how the reactions are influenced by this compartmentalization will help us gain an optimal understanding of living organisms at the molecular level and, at the same time, will offer vital clues on the behavior of simple compartmentalized systems, such as prebiotic precursors of cells and cell-inspired artificial systems. In this work we show that a reactive building block (an activated amino acid derivative) trapped in the cavity of a liposome is protected against hydrolysis and reacts nearly quantitatively with another building block, which is membrane-permeable and free in solution, to form the dipeptide. By contrast, when the activated amino acid is found outside the liposome, hydrolysis is the prevalent reaction, showing that the cavity of the liposomes promotes the formation of peptide bonds. We attribute this result to the large lipid concentration in small compartments from the point of view of a membrane-impermeable molecule. Based on this result, we show how the outcome of the reaction can be predicted as a function of the size of the compartment. The implications of these results on the behavior of biomolecules in cell compartments, abiogenesis, and the design of artificial cell-inspired systems are considered. PMID:26356087

  20. Pilot-scale pressurized base hydrolysis of HMX plastic-bonded explosives

    SciTech Connect

    Larson, S.A.; Brewer, G.R.; Harradine, D.M.; Polston, C.E.; Le, L.A.; Bishop, R.L.; Dell`Orco, P.C.; Flesner, R.L.

    1998-12-31

    A pilot-scale, pressurized, base hydrolysis reactor has been designed and its construction is nearly completed. Up to 120 L of 1--6 M NaOH aqueous solutions will convert as much as 25 kg of consolidated, explosive pieces to non-energetic compounds. Temperatures approaching 155 C in the pressurized unit will reduce reaction times significantly for the destruction of plastic-bonded explosives compared to previous atmospheric-pressure reactors. The hydrolysis effluent is then pumped into a holding tank where it is fed into a hydrothermal oxidation reactor for complete destruction to non-hazardous products. The hydrothermal unit operates at 480 C and 100 MPa and hydrogen peroxide fed into the reactor at two points will ensure complete destruction of all organic species and nitrogen-containing salts. The entire system is comprised of eight major components and is assembled on five separate and transportable skids. Following construction and preliminary testing at Los Alamos National Laboratory, the unit will be shipped to the Pantex Plant where it will be used for continuous demilitarization activities.

  1. Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group.

    PubMed

    Lim, Woon Ki; Rösgen, Jörg; Englander, S Walter

    2009-02-24

    The mechanism by which urea and guanidinium destabilize protein structure is controversial. We tested the possibility that these denaturants form hydrogen bonds with peptide groups by measuring their ability to block acid- and base-catalyzed peptide hydrogen exchange. The peptide hydrogen bonding found appears sufficient to explain the thermodynamic denaturing effect of urea. Results for guanidinium, however, are contrary to the expectation that it might H-bond. Evidently, urea and guanidinium, although structurally similar, denature proteins by different mechanisms. PMID:19196963

  2. Ion Mobility-Mass Spectrometry as a Tool for the Structural Characterization of Peptides Bearing Intramolecular Disulfide Bond(s)

    NASA Astrophysics Data System (ADS)

    Massonnet, Philippe; Haler, Jean R. N.; Upert, Gregory; Degueldre, Michel; Morsa, Denis; Smargiasso, Nicolas; Mourier, Gilles; Gilles, Nicolas; Quinton, Loïc; De Pauw, Edwin

    2016-08-01

    Disulfide bonds are post-translationnal modifications that can be crucial for the stability and the biological activities of natural peptides. Considering the importance of these disulfide bond-containing peptides, the development of new techniques in order to characterize these modifications is of great interest. For this purpose, collision cross cections (CCS) of a large data set of 118 peptides (displaying various sequences) bearing zero, one, two, or three disulfide bond(s) have been measured in this study at different charge states using ion mobility-mass spectrometry. From an experimental point of view, CCS differences (ΔCCS) between peptides bearing various numbers of disulfide bonds and peptides having no disulfide bonds have been calculated. The ΔCCS calculations have also been applied to peptides bearing two disulfide bonds but different cysteine connectivities (Cys1-Cys2/Cys3-Cys4; Cys1-Cys3/Cys2-Cys4; Cys1-Cys4/Cys2-Cys3). The effect of the replacement of a proton by a potassium adduct on a peptidic structure has also been investigated.

  3. Angiotensin I Converting Enzyme Inhibitory Peptides Obtained after In Vitro Hydrolysis of Pea (Pisum sativum var. Bajka) Globulins

    PubMed Central

    Baraniak, Barbara

    2014-01-01

    Pea seeds represent a valuable source of active compounds that may positively influence health. In this study, the pea globulins were digested in vitro under gastrointestinal condition and potentially bioaccessible angiotensin I converting enzyme (ACE) inhibitory peptides were identified. The degree of hydrolysis after pepsin, 14.42%, and pancreatin, 30.65%, were noted. The peptides with the highest ACE inhibitory properties were separated using ion exchange chromatography on DEAE-cellulose. Thirteen peptides fractions were obtained but only four showed potential antihypertensive properties. The highest inhibitory activity was determined for the fraction F8 (IC50 = 0.0014 mg/mL). This fraction was separated on Sephadex G10 and two peptide fractions were obtained. The peptides fraction (B) with the highest ACE inhibitory activity (IC50 = 0.073 mg/mL) was identified by ESI-MS/MS. The sequences of ACE inhibitory peptides were GGSGNY, DLKLP, GSSDNR, MRDLK, and HNTPSR. Based on Lineweaver-Burk plots for the fraction B, the kinetic parameters as Km, Vmax, and Ki and mode of inhibition were determined. This fraction belongs to uncompetitive inhibitor of ACE activity. The seeds of pea are the source of precursor protein, which releases the ACE inhibitory peptides as a result of enzymatic hydrolysis. PMID:25250321

  4. Hydrolysis of Surfactants Containing Ester Bonds: Modulation of Reaction Kinetics and Important Aspects of Surfactant Self-Assembly

    ERIC Educational Resources Information Center

    Lundberg, Dan; Stjerndahl, Maria

    2011-01-01

    The effects of self-assembly on the hydrolysis kinetics of surfactants that contain ester bonds are discussed. A number of examples on how reaction rates and apparent reaction orders can be modulated by changes in the conditions, including an instance of apparent zero-order kinetics, are presented. Furthermore, it is shown that the examples on…

  5. Protease-catalyzed peptide synthesis using inverse substrates: the synthesis of Pro-Xaa-bonds by trypsin.

    PubMed

    Schellenberger, V; Schellenberger, U; Jakubke, H D; Zapevalova, N P; Mitin, Y V

    1991-07-01

    Benzyloxycarbonyl-L-proline p-guanidinophenyl ester is an "inverse substrate" for trypsin; i.e., the cationic center is included in the leaving group instead of being in the acyl moiety. This substrate can be used in trypsin-catalyzed acyl-transfer reactions leading to the synthesis of Pro-Xaa peptide bonds. The reaction proceeds about 20 times slower than reaction with similar alanine-containing substrates, but the ratio between synthesis and hydrolysis is more favorable. The investigation of a series of nucleophiles led to information about the specificity of the process. Nucleophiles differing only in the P(1)'-position show an increasing acyl transfer efficiency in the order Phe < Gly < Ley < Ser < Ala < lle. C terminal elongation of the nucleophiles is of minor influence on their efficiency. The formation of an H bond between the acyl-enzyme and the nucleophile seems to play an important role in the aminolysis of the acyl-enzyme. PMID:18600766

  6. Angiotensin I-Converting Enzyme Inhibitory Peptides of Chia (Salvia hispanica) Produced by Enzymatic Hydrolysis

    PubMed Central

    Segura Campos, Maira Rubi; Peralta González, Fanny; Chel Guerrero, Luis

    2013-01-01

    Synthetic angiotensin I-converting enzyme (ACE-I) inhibitors can have undesirable side effects, while natural inhibitors have no side effects and are potential nutraceuticals. A protein-rich fraction from chia (Salvia hispanica L.) seed was hydrolyzed with an Alcalase-Flavourzyme sequential system and the hydrolysate ultrafiltered through four molecular weight cut-off membranes (1 kDa, 3 kDa, 5 kDa, and 10 kDa). ACE-I inhibitory activity was quantified in the hydrolysate and ultrafiltered fractions. The hydrolysate was extensive (DH = 51.64%) and had 58.46% ACE-inhibitory activity. Inhibition ranged from 53.84% to 69.31% in the five ultrafiltered fractions and was highest in the <1 kDa fraction (69.31%). This fraction's amino acid composition was identified and then it was purified by gel filtration chromatography and ACE-I inhibition measured in the purified fractions. Amino acid composition suggested that hydrophobic residues contributed substantially to chia peptide ACE-I inhibitory strength, probably by blocking angiotensin II production. Inhibitory activity ranged from 48.41% to 62.58% in the purified fractions, but fraction F1 (1.5–2.5 kDa) exhibited the highest inhibition (IC50 = 3.97 μg/mL; 427–455 mL elution volume). The results point out the possibility of obtaining bioactive peptides from chia proteins by means of a controlled protein hydrolysis using Alcalase-Flavourzyme sequentional system. PMID:26904588

  7. Angiotensin I-Converting Enzyme Inhibitory Peptides of Chia (Salvia hispanica) Produced by Enzymatic Hydrolysis.

    PubMed

    Segura Campos, Maira Rubi; Peralta González, Fanny; Chel Guerrero, Luis; Betancur Ancona, David

    2013-01-01

    Synthetic angiotensin I-converting enzyme (ACE-I) inhibitors can have undesirable side effects, while natural inhibitors have no side effects and are potential nutraceuticals. A protein-rich fraction from chia (Salvia hispanica L.) seed was hydrolyzed with an Alcalase-Flavourzyme sequential system and the hydrolysate ultrafiltered through four molecular weight cut-off membranes (1 kDa, 3 kDa, 5 kDa, and 10 kDa). ACE-I inhibitory activity was quantified in the hydrolysate and ultrafiltered fractions. The hydrolysate was extensive (DH = 51.64%) and had 58.46% ACE-inhibitory activity. Inhibition ranged from 53.84% to 69.31% in the five ultrafiltered fractions and was highest in the <1 kDa fraction (69.31%). This fraction's amino acid composition was identified and then it was purified by gel filtration chromatography and ACE-I inhibition measured in the purified fractions. Amino acid composition suggested that hydrophobic residues contributed substantially to chia peptide ACE-I inhibitory strength, probably by blocking angiotensin II production. Inhibitory activity ranged from 48.41% to 62.58% in the purified fractions, but fraction F1 (1.5-2.5 kDa) exhibited the highest inhibition (IC50 = 3.97 μg/mL; 427-455 mL elution volume). The results point out the possibility of obtaining bioactive peptides from chia proteins by means of a controlled protein hydrolysis using Alcalase-Flavourzyme sequentional system. PMID:26904588

  8. Heterologous expression of five disulfide-bonded insecticidal spider peptides.

    PubMed

    Estrada, Georgina; Silva, Anita O; Villegas, Elba; Ortiz, Ernesto; Beirão, Paulo S L; Corzo, Gerardo

    2016-09-01

    The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from the spider Oxyopes lineatus were cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage region. These two recombinant vectors were transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The product of each gene was named HisrOxyTx1 or HisrOxyTx2, and the protein expression was ca 14 and 6 mg/L of culture medium, respectively. Either recombinant toxin HisrOxyTx1 or HisrOxyTx2 were found exclusively in inclusion bodies, which were solubilized using a chaotropic agent, and then, purified using affinity chromatography and reverse-phase HPLC (RP-HPLC). The HisrOxyTx1 and HisrOxyTx2 products, obtained from the affinity chromatographic step, showed several peptide fractions having the same molecular mass of 9913.1 and 8030.1 Da, respectively, indicating that both HisrOxyTx1 and HisrOxyTx2 were oxidized forming several distinct disulfide bridge arrangements. The isoforms of both HisrOxyTx1 and HisrOxyTx2 after DTT reduction eluted from the column as a single protein component of 9923 and 8040 Da, respectively. In vitro folding of either HisrOxyTx1 or HisrOxyTx2 yielded single oxidized components, which were cleaved independently by the proteolytic enzyme Factor Xa to give the recombinant peptides rOxyTx1 and rOxyTx2. The experimental molecular masses of rOxyTx1 and rOxyTx2 were 8059.0 and 6176.4 Da, respectively, which agree with their expected theoretical masses. The recombinant peptides rOxyTx1 and rOxyTx2 showed lower but comparable toxicity to the native toxins when injected into lepidopteran larvae; furthermore, rOxyTx1 was able to inhibit calcium ion currents on dorsal unpaired median (DUM) neurons from Periplaneta americana. PMID:27263806

  9. Novel dehydrogenase catalyzes oxidative hydrolysis of carbon-nitrogen double bonds for hydrazone degradation.

    PubMed

    Itoh, Hideomi; Suzuta, Tetsuya; Hoshino, Takayuki; Takaya, Naoki

    2008-02-29

    Hydrazines and their derivatives are versatile artificial and natural compounds that are metabolized by elusive biological systems. Here we identified microorganisms that assimilate hydrazones and isolated the yeast, Candida palmioleophila MK883. When cultured with adipic acid bis(ethylidene hydrazide) as the sole source of carbon, C. palmioleophila MK883 degraded hydrazones and accumulated adipic acid dihydrazide. Cytosolic NAD+- or NADP+-dependent hydrazone dehydrogenase (Hdh) activity was detectable under these conditions. The production of Hdh was inducible by adipic acid bis(ethylidene hydrazide) and the hydrazone, varelic acid ethylidene hydrazide, under the control of carbon catabolite repression. Purified Hdh oxidized and hydrated the C=N double bond of acetaldehyde hydrazones by reducing NAD+ or NADP+ to produce relevant hydrazides and acetate, the latter of which the yeast assimilated. The deduced amino acid sequence revealed that Hdh belongs to the aldehyde dehydrogenase (Aldh) superfamily. Kinetic and mutagenesis studies showed that Hdh formed a ternary complex with the substrates and that conserved Cys is essential for the activity. The mechanism of Hdh is similar to that of Aldh, except that it catalyzed oxidative hydrolysis of hydrazones that requires adding a water molecule to the reaction catalyzed by conventional Aldh. Surprisingly, both Hdh and Aldh from baker's yeast (Ald4p) catalyzed the Hdh reaction as well as aldehyde oxidation. Our findings are unique in that we discovered a biological mechanism for hydrazone utilization and a novel function of proteins in the Aldh family that act on C=N compounds. PMID:18096698

  10. Production of ACE inhibitory peptides from sweet sorghum grain protein using alcalase: Hydrolysis kinetic, purification and molecular docking study.

    PubMed

    Wu, Qiongying; Du, Jinjuan; Jia, Junqiang; Kuang, Cong

    2016-05-15

    In this study, sweet sorghum grain protein (SSGP) was hydrolyzed using alcalase yielding ACE inhibitory peptides. A kinetic model was proposed to describe the enzymolysis process of SSGP. The kinetic parameters, a and b, were determined according to experimental data. It was found that the model was reliable to describe the kinetic behaviour for SSGP hydrolysis by alcalase. After hydrolysis, the SSGP hydrolysate with DH of 19% exhibited the strongest ACE inhibitory activity and the hydrolysate was then used to isolate ACE inhibitory peptides. A novel ACE inhibitory peptide was successfully purified from this hydrolysate by ultrafiltration, ion exchange chromatography, gel filtration chromatography, and reversed-phased high performance liquid chromatography (RP-HPLC). The amino acid sequence of the purified peptide was identified as Thr-Leu-Ser (IC50=102.1 μM). The molecular docking studies revealed that the ACE inhibition of the tripeptide was mainly attributed to its C-terminal Ser, which can effectively interact with the S1 and S2 pockets of ACE. Our studies suggest that the tripeptide from the SSGP hydrolysate can be utilized to develop functional food ingredients or pharmaceuticals for prevention of hypertension. PMID:26775955

  11. Slow peptide bond formation by proline and other N-alkylamino acids in translation.

    PubMed

    Pavlov, Michael Y; Watts, Richard E; Tan, Zhongping; Cornish, Virginia W; Ehrenberg, Måns; Forster, Anthony C

    2009-01-01

    Proteins are made from 19 aa and, curiously, one N-alkylamino acid ("imino acid"), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3-6 times slower than Phe incorporation from Phe-tRNA(Phe) depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNA(Pro) isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the "tRNA abundance" hypothesis. PMID:19104062

  12. Resynthesis of reactive site peptide bond and temporary inhibition of Streptomyces metalloproteinase inhibitor.

    PubMed

    Seeram, S S; Hiraga, K; Oda, K

    1997-10-01

    Streptomyces metalloproteinase inhibitor (SMPI) is a small proteinaceous inhibitor which inhibits metalloproteinases such as thermolysin (Ki =1.14 x 10(-10) M). When incubated with the enzyme, it is gradually hydrolyzed at the Cys64-Val65 peptide bond, which was identified as the reactive site by mutational analysis. To achieve a further understanding of the inhibition mechanism, we attempted to resynthesize the cleaved reactive site by using the enzyme catalytic action. The native inhibitor was resynthesized from the modified inhibitor (Ki =2.18 x 10(-8) M) by incubation with a catalytic amount of thermolysin under the same conditions as used for hydrolysis (pH 7.5, 25 degrees C), suggesting that SMPI follows the standard mechanism of inhibition of serine proteinase inhibitors. Temporary inhibition was observed when the native inhibitor and thermolysin were incubated at a 1:100 (mol/mol) enzyme-inhibitor ratio at 37 degrees C. SMPI showed temporary inhibition towards all the enzymes it inhibited. The inhibitory spectrum of SMPI was analyzed with various metalloproteinases based on the Ki values and limited proteolysis patterns. Pseudomonas elastase and Streptomyces griseus metalloproteinase II formed more stable complexes and showed much lower Ki values (approximately 2 pM) than thermolysin. In the limited proteolysis experiments weak inhibitors were degraded by the enzymes. SMPI did not inhibit almelysin, Streptomyces caespitosus neutral proteinase or matrix metalloproteinases. SMPI specifically inhibits metalloproteinases which are sensitive to phosphoramidon. PMID:9399583

  13. Selectivity of peptide bond dissociation on excitation of a core electron: Effects of a phenyl group

    NASA Astrophysics Data System (ADS)

    Tsai, Cheng-Cheng; Chen, Jien-Lian; Hu, Wei-Ping; Lin, Yi-Shiue; Lin, Huei-Ru; Lee, Tsai-Yun; Lee, Yuan T.; Ni, Chi-Kung; Liu, Chen-Lin

    2016-09-01

    The selective dissociation of a peptide bond upon excitation of a core electron in acetanilide and N-benzylacetamide was investigated. The total-ion-yield near-edge X-ray absorption fine structure spectra were recorded and compared with the predictions from time-dependent density functional theory. The branching ratios for the dissociation of a peptide bond are observed as 16-34% which is quite significant. This study explores the core-excitation, the X-ray photodissociation pathways, and the theoretical explanation of the NEXAFS spectra of organic molecules containing both a peptide bond and a phenyl group.

  14. Complete amino acid analysis of peptides and proteins after hydrolysis by a mixture of Sepharose-bound peptidases

    PubMed Central

    Bennett, H. P. J.; Elliott, D. F.; Evans, B. E.; Lowry, P. J.; McMartin, C.

    1972-01-01

    Incubation with a mixture of Sepharose-bound peptidases was shown to result in the quantitative release of amino acids from certain peptides and S-aminoethylated proteins. Subtraction of the low background values of amino acids generated by the enzymes enables amino acid ratios of corticotrophin-(1–24)-tetracosapeptide to be determined with a standard deviation on repeat digestions of 3–5%. Good values were obtained for amino acids that are completely or partially destroyed on acid hydrolysis, i.e. tryptophan, tyrosine, serine, asparagine and glutamine. Experiments with peptides containing d-amino acids showed that the enzyme mixture is stereospecific and could therefore be used to detect the presence of d-residues in peptides. The enzyme mixture completely hydrolyses peptide fragments obtained after Edman degradation and should therefore be useful for determining sequences of peptides containing acid-labile amino acid residues. The activities of the bound enzymes were unaltered over a period of 7 months and they provide a simple, reproducible procedure for the quantitative determination of amino acids in peptides and proteins containing l-amino acids. PMID:4349115

  15. Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities.

    PubMed

    Thomas, N E; Bramson, H N; Miller, W T; Kaiser, E T

    1987-07-14

    As part of a search for peptides that have specificity for selected protein kinases, the possibility that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) recognizes the hydrogen-bonding potential of its peptide substrates was investigated. A-Kinase catalyzes the phosphorylation of five N alpha-methylated and four depsipeptide derivatives of Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) at rates that differ by at least 7 orders of magnitude. These peptide 1 analogues each lack the ability to donate a hydrogen bond at selected positions in the peptide chain. If a particular amide hydrogen of a peptide amide is involved in hydrogen bonding, which is important for enzyme recognition, the prediction is that peptides which contain an ester or a N-methylated bond at that position in peptide 1 will be comparatively poor substrates. In contrast, if a depsipeptide has a reactivity comparable to that of peptide 1 but the analogous N-methylated peptide has a poor reactivity with A-kinase, the result might indicate that the N-methyl group causes unfavorable steric effects. The depsipeptide that lacks a Leu6 amide proton is a good substrate for A-kinase, but the corresponding N-methylated peptide is phosphorylated far less efficiently. This result and others presented in this paper suggest that although enzyme-substrate hydrogen bonding may play some role in A-kinase catalysis of phosphoryl group transfer, other explanations are necessary to account for the relative reactivities of N alpha-methylated and depsi-containing peptide 1 analogues.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3663600

  16. Antioxidative Peptides Derived from Enzyme Hydrolysis of Bone Collagen after Microwave Assisted Acid Pre-Treatment and Nitrogen Protection

    PubMed Central

    Lin, Yun-Jian; Le, Guo-Wei; Wang, Jie-Yun; Li, Ya-Xin; Shi, Yong-Hui; Sun, Jin

    2010-01-01

    This study focused on the preparation method of antioxidant peptides by enzymatic hydrolysis of bone collagen after microwave assisted acid pre-treatment and nitrogen protection. Phosphoric acid showed the highest ability of hydrolysis among the four other acids tested (hydrochloric acid, sulfuric acid and/or citric acid). The highest degree of hydrolysis (DH) was 9.5% using 4 mol/L phosphoric acid with a ratio of 1:6 under a microwave intensity of 510 W for 240 s. Neutral proteinase gave higher DH among the four protease tested (Acid protease, neutral protease, Alcalase and papain), with an optimum condition of: (1) ratio of enzyme and substrate, 4760 U/g; (2) concentration of substrate, 4%; (3) reaction temperature, 55 °C and (4) pH 7.0. At 4 h, DH increased significantly (P < 0.01) under nitrogen protection compared with normal microwave assisted acid pre-treatment hydrolysis conditions. The antioxidant ability of the hydrolysate increased and reached its maximum value at 3 h; however DH decreased dramatically after 3 h. Microwave assisted acid pre-treatment and nitrogen protection could be a quick preparatory method for hydrolyzing bone collagen. PMID:21151439

  17. Applications of 2D IR spectroscopy to peptides, proteins, and hydrogen-bond dynamics

    PubMed Central

    Kim, Yung Sam; Hochstrasser, Robin M.

    2010-01-01

    Following a survey of 2D IR principles this Feature Article describes recent experiments on the hydrogen-bond dynamics of small ions, amide-I modes, nitrile probes, peptides, reverse transcriptase inhibitors, and amyloid fibrils. PMID:19351162

  18. Identification of the Major ACE-Inhibitory Peptides Produced by Enzymatic Hydrolysis of a Protein Concentrate from Cuttlefish Wastewater

    PubMed Central

    Rodríguez Amado, Isabel; Vázquez, José Antonio; González, Pilar; Esteban-Fernández, Diego; Carrera, Mónica; Piñeiro, Carmen

    2014-01-01

    The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (IC50 = 76.8 ± 15.2 μg mL−1) after 8 h of proteolysis. Sequential ultrafiltration of the 8 h hydrolysate with molecular weight cut-off (MWCO) membranes of 10 and 1 kDa resulted in the increased activity of each permeate, with a final IC50 value of 58.4 ± 4.6 μg mL−1. Permeate containing peptides lower than 1 kDa was separated by reversed-phase high performance liquid chromatography (RP-HPLC). Four fractions (A–D) with potent ACE inhibitory activity were isolated and their main peptides identified using high performance liquid chromatography coupled to an electrospray ion trap Fourier transform ion cyclotron resonance-mass spectrometer (HPLC-ESI-IT-FTICR) followed by comparison with databases and de novo sequencing. The amino acid sequences of the identified peptides contained at least one hydrophobic and/or a proline together with positively charged residues in at least one of the three C-terminal positions. The IC50 values of the fractions ranged from 1.92 to 8.83 μg mL−1, however this study fails to identify which of these peptides are ultimately responsible for the potent antihypertensive activity of these fractions. PMID:24619242

  19. Biologically active peptides obtained by enzymatic hydrolysis of Adzuki bean seeds.

    PubMed

    Durak, Agata; Baraniak, Barbara; Jakubczyk, Anna; Świeca, Michał

    2013-12-01

    This study investigated the antioxidant and antihypertensive activities of peptides obtained from protein fractions of Adzuki bean seeds. Peptides were obtained by the use of hydrolytic enzymes in vitro under gastrointestinal conditions. A determination was made of the activity of the peptide inhibitors of the angiotensin I converting enzyme (ACE), and the antiradical and ion chelating activity of peptides from different protein fractions. The highest peptide levels after the absorption process (<7 kDa) were noted in the albumin fraction (50.69 μg/ml). Furthermore, it was found that peptides from the prolamin fraction were characterised by the highest antiradical activity and ACE inhibitory activity (IC50=0.17 mg/ml). Peptides obtained from the globulin fraction showed the highest ability to chelate iron ions, and peptides from the glutelin fraction were characterised as being the most effective in the chelation of copper ions. PMID:23870945

  20. Enzymatic hydrolysis of ovomucin and the functional and structural characteristics of peptides in the hydrolysates.

    PubMed

    Abeyrathne, E D N S; Lee, H Y; Jo, C; Suh, J W; Ahn, D U

    2016-02-01

    Ovomucin was hydrolyzed using enzymes or by heating under alkaline conditions (pH 12.0), and the functional, structural and compositional characteristics of the peptides in the hydrolysates were determined. Among the treatments, heating at 100 °C for 15 min under alkaline conditions (OM) produced peptides with the highest iron-binding and antioxidant capacities. Ovomucin hydrolyzed with papain (OMPa) or alcalase (OMAl) produced peptides with high ACE-inhibitory activity. The mass spectrometry analysis indicated that most of the peptides from OMPa were <2 kDa, but peptides from OMTr and OM were >2 kDa. OMAl hydrolyzed ovomucin almost completely and no peptides within 700-5000 Da were found in the hydrolasate. The results indicated that the number and size of peptides were closely related to the functionality of the hydrolysates. Considering the time, cost and activities of the hydrolysates, OM was the best treatment for hydrolyzing ovomucin to produce functional peptides. PMID:26304326

  1. Efficient Covalent Bond Formation in Gas-Phase Peptide-Peptide Ion Complexes with the Photoleucine Stapler.

    PubMed

    Shaffer, Christopher J; Andrikopoulos, Prokopis C; Řezáč, Jan; Rulíšek, Lubomír; Tureček, František

    2016-04-01

    Noncovalent complexes of hydrophobic peptides GLLLG and GLLLK with photoleucine (L*) tagged peptides G(L* n L m )K (n = 1,3, m = 2,0) were generated as singly charged ions in the gas phase and probed by photodissociation at 355 nm. Carbene intermediates produced by photodissociative loss of N2 from the L* diazirine rings underwent insertion into X-H bonds of the target peptide moiety, forming covalent adducts with yields reaching 30%. Gas-phase sequencing of the covalent adducts revealed preferred bond formation at the C-terminal residue of the target peptide. Site-selective carbene insertion was achieved by placing the L* residue in different positions along the photopeptide chain, and the residues in the target peptide undergoing carbene insertion were identified by gas-phase ion sequencing that was aided by specific (13)C labeling. Density functional theory calculations indicated that noncovalent binding to GL*L*L*K resulted in substantial changes of the (GLLLK + H)(+) ground state conformation. The peptide moieties in [GL*L*LK + GLLLK + H](+) ion complexes were held together by hydrogen bonds, whereas dispersion interactions of the nonpolar groups were only secondary in ground-state 0 K structures. Born-Oppenheimer molecular dynamics for 100 ps trajectories of several different conformers at the 310 K laboratory temperature showed that noncovalent complexes developed multiple, residue-specific contacts between the diazirine carbons and GLLLK residues. The calculations pointed to the substantial fluidity of the nonpolar side chains in the complexes. Diazirine photochemistry in combination with Born-Oppenheimer molecular dynamics is a promising tool for investigations of peptide-peptide ion interactions in the gas phase. Graphical Abstract ᅟ. PMID:26817657

  2. Efficient Covalent Bond Formation in Gas-Phase Peptide-Peptide Ion Complexes with the Photoleucine Stapler

    NASA Astrophysics Data System (ADS)

    Shaffer, Christopher J.; Andrikopoulos, Prokopis C.; Řezáč, Jan; Rulíšek, Lubomír; Tureček, František

    2016-04-01

    Noncovalent complexes of hydrophobic peptides GLLLG and GLLLK with photoleucine (L*) tagged peptides G(L* n L m )K (n = 1,3, m = 2,0) were generated as singly charged ions in the gas phase and probed by photodissociation at 355 nm. Carbene intermediates produced by photodissociative loss of N2 from the L* diazirine rings underwent insertion into X-H bonds of the target peptide moiety, forming covalent adducts with yields reaching 30%. Gas-phase sequencing of the covalent adducts revealed preferred bond formation at the C-terminal residue of the target peptide. Site-selective carbene insertion was achieved by placing the L* residue in different positions along the photopeptide chain, and the residues in the target peptide undergoing carbene insertion were identified by gas-phase ion sequencing that was aided by specific 13C labeling. Density functional theory calculations indicated that noncovalent binding to GL*L*L*K resulted in substantial changes of the (GLLLK + H)+ ground state conformation. The peptide moieties in [GL*L*LK + GLLLK + H]+ ion complexes were held together by hydrogen bonds, whereas dispersion interactions of the nonpolar groups were only secondary in ground-state 0 K structures. Born-Oppenheimer molecular dynamics for 100 ps trajectories of several different conformers at the 310 K laboratory temperature showed that noncovalent complexes developed multiple, residue-specific contacts between the diazirine carbons and GLLLK residues. The calculations pointed to the substantial fluidity of the nonpolar side chains in the complexes. Diazirine photochemistry in combination with Born-Oppenheimer molecular dynamics is a promising tool for investigations of peptide-peptide ion interactions in the gas phase.

  3. Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

    PubMed

    Chufan, Eduardo E; Kapoor, Khyati; Ambudkar, Suresh V

    2016-02-01

    P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp. PMID:26686578

  4. Hydrolysis of whey protein isolate with Bacillus licheniformis protease: aggregating capacities of peptide fractions.

    PubMed

    Creusot, Nathalie; Gruppen, Harry

    2008-11-12

    In a previous study, peptides aggregating at pH 7.0 derived from a whey protein hydrolysate made with Bacillus licheniformis protease were fractionated and identified. The objective of the present work was to investigate the solubility of the fractionated aggregating peptides, as a function of concentration, and their aggregating capacities toward added intact proteins. The amount of aggregated material and the composition of the aggregates obtained were measured by nitrogen concentration and size exclusion chromatography, respectively. The results showed that of the four fractions obtained from the aggregating peptides, two were insoluble, while the other two consisted of 1:1 mixture of low and high solubility peptides. Therefore, insoluble peptides coaggregated, assumedly via hydrophobic interactions, other relatively more soluble peptides. It was also shown that aggregating peptides could aggregate intact protein nonspecifically since the same peptides were involved in the aggregation of whey proteins, beta-casein, and bovine serum albumin. Both insoluble and partly insoluble peptides were required for the aggregation of intact protein. These results are of interest for the applications of protein hydrolysates, as mixtures of intact protein and peptides are often present in these applications. PMID:18922012

  5. Antimicrobial activities of amphiphilic peptides covalently bonded to a water-insoluble resin.

    PubMed Central

    Haynie, S L; Crum, G A; Doele, B A

    1995-01-01

    A series of polymer-bound antimicrobial peptides was prepared, and the peptides were tested for their antimicrobial activities. The immobilized peptides were prepared by a strategy that used solid-phase peptide synthesis that linked the carboxy-terminal amino acid with an ethylenediamine-modified polyamide resin (PepsynK). The acid-stable, permanent amide bond between the support and the nascent peptide renders the peptide resistant to cleavage from the support during the final acid-catalyzed deprotection step in the synthesis. Select immobilized peptides containing amino acid sequences that ranged from the naturally occurring magainin to simpler synthetic sequences with idealized secondary structures were excellent antimicrobial agents against several organisms. The immobilized peptides typically reduced the number of viable cells by > or = 5 log units. We show that the reduction in cell numbers cannot be explained by the action of a soluble component. We observed no leached or hydrolyzed peptide from the resin, nor did we observe any antimicrobial activity in soluble extracts from the immobilized peptide. The immobilized peptides were washed and reused for repeated microbial contact and killing. These results suggest that the surface actions by magainins and structurally related antimicrobial peptides are sufficient for their lethal activities. PMID:7726486

  6. Slow peptide bond formation by proline and other N-alkylamino acids in translation

    PubMed Central

    Pavlov, Michael Y.; Watts, Richard E.; Tan, Zhongping; Cornish, Virginia W.; Ehrenberg, Måns; Forster, Anthony C.

    2009-01-01

    Proteins are made from 19 aa and, curiously, one N-alkylamino acid (“imino acid”), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3–6 times slower than Phe incorporation from Phe-tRNAPhe depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNAPro isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the “tRNA abundance” hypothesis. PMID:19104062

  7. Estimation of peptide N-Cα bond cleavage efficiency during MALDI-ISD using a cyclic peptide.

    PubMed

    Asakawa, Daiki; Smargiasso, Nicolas; De Pauw, Edwin

    2016-05-01

    Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) induces N-Cα bond cleavage via hydrogen transfer from the matrix to the peptide backbone, which produces a c'/z• fragment pair. Subsequently, the z• generates z' and [z + matrix] fragments via further radical reactions because of the low stability of the z•. In the present study, we investigated MALDI-ISD of a cyclic peptide. The N-Cα bond cleavage in the cyclic peptide by MALDI-ISD produced the hydrogen-abundant peptide radical [M + 2H](+) • with a radical site on the α-carbon atom, which then reacted with the matrix to give [M + 3H](+) and [M + H + matrix](+) . For 1,5-diaminonaphthalene (1,5-DAN) adducts with z fragments, post-source decay of [M + H + 1,5-DAN](+) generated from the cyclic peptide showed predominant loss of an amino acid with 1,5-DAN. Additionally, MALDI-ISD with Fourier transform-ion cyclotron resonance mass spectrometry allowed for the detection of both [M + 3H](+) and [M + H](+) with two (13) C atoms. These results strongly suggested that [M + 3H](+) and [M + H + 1,5-DAN](+) were formed by N-Cα bond cleavage with further radical reactions. As a consequence, the cleavage efficiency of the N-Cα bond during MALDI-ISD could be estimated by the ratio of the intensity of [M + H](+) and [M + 3H](+) in the Fourier transform-ion cyclotron resonance spectrum. Because the reduction efficiency of a matrix for the cyclic peptide cyclo(Arg-Gly-Asp-D-Phe-Val) was correlated to its tendency to cleave the N-Cα bond in linear peptides, the present method could allow the evaluation of the efficiency of N-Cα bond cleavage for MALDI matrix development. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27194516

  8. Catalysis of hydrolysis and nucleophilic substitution at the P-N bond of phosphoimidazolide-activated nucleotides in phosphate buffers

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Rosenbach, M. T.

    1991-01-01

    Phosphoimidazolide-activated derivatives of guanosine and cytidine 5'-monophosphates, henceforth called ImpN's, exhibit enhanced rates of degradation in the presence of aqueous inorganic phosphate in the range 4.0 < or = pH < or = 8.6. This degradation is been attributed to (i) nucleophilic substitution of the imidazolide and (ii) catalysis of the P-N bond hydrolysis by phosphate. The first reaction results in the formation of nucleoside 5'-diphosphate and the second in nucleoside 5'-monophosphate. Analysis of the observed rates as well as the product ratios as a function of pH and phosphate concentration allow distinction between various mechanistic possibilities. The results show that both H2PO4- and HPO4(2-) participate in both hydrolysis and nucleophilic substitution. Statistically corrected biomolecular rate constants indicate that the dianion is 4 times more effective as a general base than the monoanion, and 8 times more effective as nucleophile. The low Bronsted value beta = 0.15 calculated for these phosphate species, presumed to act as general bases in facilitating water attack, is consistent with the fact that catalysis of the hydrolysis of the P-N bond in ImpN's has not been detected before. The beta nuc = 0.35 calculated for water, H2PO4-, HPO4(2-), and hydroxide acting as nucleophiles indicates a more associative transition state for nucleotidyl (O2POR- with R = nucleoside) transfers than that observed for phosphoryl (PO3(2-)) transfers (beta nuc = 0.25). With respect to the stability/reactivity of ImpN's under prebiotic conditions, our study shows that these materials would not suffer additional degradation due to inorganic phosphate, assuming the concentrations of phosphate, Pi, on prebiotic Earth were similar to those in the present oceans ([Pi] approximately 2.25 micromoles).

  9. Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts

    PubMed Central

    Chou, Ming-Lun; Chu, Chiung-Chih; Chen, Lih-Jen; Akita, Mitsuru; Li, Hsou-min

    2006-01-01

    Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing. The Tic40 C-terminal domain, which is homologous to the C terminus of cochaperones Sti1p/Hop and Hip but with no known function, stimulates adenosine triphosphate hydrolysis by Hsp93. Hsp93 dissociates from Tic40 in the presence of adenosine diphosphate, suggesting that Tic40 functions as an adenosine triphosphatase activation protein for Hsp93. Our data suggest that chloroplasts have evolved the Tic40 cochaperone to increase the efficiency of precursor processing and translocation. PMID:17158958

  10. Disulphide bonds in wheat gluten: cystine peptides derived from gluten proteins following peptic and thermolytic digestion.

    PubMed

    Keck, B; Köhler, P; Wieser, H

    1995-06-01

    Gluten from the wheat variety Rektor was extracted with 70% aqueous ethanol. The insoluble portion (whole glutenin) was partially hydrolysed with trypsin at pH 6.5 and separated on a Sephadex G25 column. The high molecular weight fraction 1 was further hydrolysed with pepsin at pH 2.0. To remove low molecular weight proteins, a portion of whole glutenin was extracted with dilute acetic acid. The residue (enriched glutenin), which contained mostly LMW and HMW subunits of glutenin, was hydrolysed with thermolysin at pH 6.5. The peptic and tryptic hydrolysates were separated on a Sephadex G25 column and the peptide fractions with the highest cystine content were separated further by reversed-phase high-performance liquid chromatography (RP-HPLC). Cystine peptides were detected by differential chromatography (RP-HPLC prior to and after reduction of disulphide bonds) and then isolated by preparative RP-HPLC. After reduction, cysteine peptides were alkylated and analysed for their amino acid sequence. Altogether, 19 cystine peptides were characterized and assigned to known sequences of gluten proteins; 16 peptides confirmed the positions of disulphide bonds present in LMW subunits and gamma-gliadins, as described previously. For the first time, a cystine peptide has been isolated, representing an intermolecular disulphide bond between the y-type of HMW and LMW subunits. Furthermore, a cystine peptide was assigned to gamma-gliadins; thus, all cysteine residues of gamma-gliadins are documented by at least one cystine peptide. One peptide analysed came from the alpha-amylase inhibitor CM 16. Altogether the results indicate that the intramolecular linkages of gluten proteins are not formed at random, but are strongly directed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7668061

  11. Purification and identification of lipolysis-stimulating peptides derived from enzymatic hydrolysis of soy protein.

    PubMed

    Tsou, May-June; Kao, Fuh-Juin; Lu, Hsi-Chi; Kao, Hao-Chun; Chiang, Wen-Dee

    2013-06-01

    The aim of this study was to purify and identify lipolysis-stimulating peptides derived from Flavourzyme®-soy protein isolate (SPI) hydrolysate (F-SPIH). Glycerol release was employed as a marker for lipolysis in 3T3-L1 adipocytes. A higher glycerol release represents a better lipolysis-stimulating activity. The peptide fraction with highest glycerol release obtained from F-SPIH fractionated by sequential ultrafiltration membranes was further purified using gel filtration chromatography and two steps of reverse-phase high-performance liquid chromatography. The peptides were identified using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Three lipolysis-stimulating peptides were obtained, and the amino acid sequences were ILL, LLL and VHVV, respectively. The in vitro effect of gastrointestinal proteases on lipolysis-stimulating activity of synthetic ILL, LLL and VHVV, respectively, was also investigated. The result suggested that the gastrointestinal protease did not affect lipolysis-stimulating activity of the three novel peptides, which reveals their potential to act as anti-obesity ingredients. PMID:23411267

  12. Acid Hydrolysis and Molecular Density of Phytoglycogen and Liver Glycogen Helps Understand the Bonding in Glycogen α (Composite) Particles

    PubMed Central

    Powell, Prudence O.; Sullivan, Mitchell A.; Sheehy, Joshua J.; Schulz, Benjamin L.; Warren, Frederick J.; Gilbert, Robert G.

    2015-01-01

    Phytoglycogen (from certain mutant plants) and animal glycogen are highly branched glucose polymers with similarities in structural features and molecular size range. Both appear to form composite α particles from smaller β particles. The molecular size distribution of liver glycogen is bimodal, with distinct α and β components, while that of phytoglycogen is monomodal. This study aims to enhance our understanding of the nature of the link between liver-glycogen β particles resulting in the formation of large α particles. It examines the time evolution of the size distribution of these molecules during acid hydrolysis, and the size dependence of the molecular density of both glucans. The monomodal distribution of phytoglycogen decreases uniformly in time with hydrolysis, while with glycogen, the large particles degrade significantly more quickly. The size dependence of the molecular density shows qualitatively different shapes for these two types of molecules. The data, combined with a quantitative model for the evolution of the distribution during degradation, suggest that the bonding between β into α particles is different between phytoglycogen and liver glycogen, with the formation of a glycosidic linkage for phytoglycogen and a covalent or strong non-covalent linkage, most probably involving a protein, for glycogen as most likely. This finding is of importance for diabetes, where α-particle structure is impaired. PMID:25799321

  13. Enhancement of peptide bond formation by polyribonucleotides on clay surfaces in fluctuating environments

    NASA Technical Reports Server (NTRS)

    White, D. H.; Erickson, J. C.

    1981-01-01

    The selective effects of polyribonucleotides on the formation of glycine peptide bonds in glycine on clay surfaces are investigated as a model for a template mechanism for the effects of polynucleotides on peptide bond formation. Free oligoglycine yields were determined for the cycling reaction of glycine in the presence and absence of clay and polyribonucleotides or polydeoxyribonucleotides. The polyribonucleotides are observed to lead to increases of up to fourfold increases in oligoglycine formed, with greater enhancements for poly-G nucleotides than for poly-A, poly-U and poly-C, indicating a codonic bias. Polydeoxyribonucleotides are found to provide no enhancement in peptide formation rates, and yields were also greatly reduced in the absence of clay. A mechanism for peptide synthesis is proposed which involves the activation of glycine on the clay surface, followed by the formation of esters between glycine and the 2-prime OH groups of the polyribonucleotide and peptide bonds between adjacent amino acyl esters. It is pointed out that if this mechanism is correct, it may provide a basis for a direct template translation process, which would produce a singlet genetic code.

  14. 1,2,3-Triazole Bridge as Conformational Constrain in β-Hairpin Peptides: Analysis of Hydrogen-Bonded Positions.

    PubMed

    Celentano, V; Diana, D; Di Salvo, C; De Rosa, L; Romanelli, A; Fattorusso, R; D'Andrea, L D

    2016-04-11

    Conformational constrained β-hairpin peptides are useful tool to modulate protein-protein interactions. A triazole bridge in hydrogen-bonded positions between two antiparallel strands induces a conformational stabilization of the β-hairpin peptide. The entity of the stability of the β-hairpin peptide depends on the length of the bridge. PMID:26938670

  15. A structural view on the mechanism of the ribosome-catalyzed peptide bond formation

    PubMed Central

    Simonović, Miljan; Steitz, Thomas A.

    2009-01-01

    The ribosome is a large ribonucleoprotein particle that translates zgenetic information encoded in mRNA into specific proteins. Its highly conserved active site, the peptidyl-transferase center (PTC), is located on the large (50S) ribosomal subunit and is comprised solely of rRNA, which makes the ribosome the only natural ribozyme with polymerase activity. The last decade witnessed a rapid accumulation of atomic-resolution structural data on both ribosomal subunits as well as on the entire ribosome. This has allowed studies on the mechanism of peptide bond formation at a level of detail that surpasses that for the classical protein enzymes. A current understanding of the mechanism of the ribosome-catalyzed peptide bond formation is the focus of this review. Implications on the mechanism of peptide release are discussed as well. PMID:19595805

  16. Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

    SciTech Connect

    Agarwal, Vinayak; Borisova, Svetlana A.; Metcalf, William W.; van der Donk, Wilfred A.; Nair, Satish K.

    2011-12-22

    Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 {angstrom} resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases but with notable differences, such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional cocrystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently bound transition state mimic provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily.

  17. D-β-aspartyl residue exhibiting uncommon high resistance to spontaneous peptide bond cleavage

    PubMed Central

    Aki, Kenzo; Okamura, Emiko

    2016-01-01

    Although L-amino acids were selected as main constituents of peptides and proteins during chemical evolution, D-aspartyl (Asp) residue is found in a variety of living tissues. In particular, D-β-Asp is thought to be stable than any other Asp isomers, and this could be a reason for gradual accumulation in abnormal proteins and peptides to modify their structures and functions. It is predicted that D-β-Asp shows high resistance to biomolecular reactions. For instance, less reactivity of D-β-Asp is expected to bond cleavage, although such information has not been provided yet. In this work, the spontaneous peptide bond cleavage was compared between Asp isomers, by applying real-time solution-state NMR to eye lens αΑ-crystallin 51–60 fragment, S51LFRTVLD58SG60 and αΒ-crystallin 61–67 analog, F61D62TGLSG67 consisting of L-α- and D-β-Asp 58 and 62, respectively. Kinetic analysis showed how tough the uncommon D-β-Asp residue was against the peptide bond cleavage as compared to natural L-α-Asp. Differences in pKa and conformation between L-α- and D-β-Asp side chains were plausible factors to determine reactivity of Asp isomers. The present study, for the first time, provides a rationale to explain less reactivity of D-β-Asp to allow abnormal accumulation. PMID:26876027

  18. D-β-aspartyl residue exhibiting uncommon high resistance to spontaneous peptide bond cleavage

    NASA Astrophysics Data System (ADS)

    Aki, Kenzo; Okamura, Emiko

    2016-02-01

    Although L-amino acids were selected as main constituents of peptides and proteins during chemical evolution, D-aspartyl (Asp) residue is found in a variety of living tissues. In particular, D-β-Asp is thought to be stable than any other Asp isomers, and this could be a reason for gradual accumulation in abnormal proteins and peptides to modify their structures and functions. It is predicted that D-β-Asp shows high resistance to biomolecular reactions. For instance, less reactivity of D-β-Asp is expected to bond cleavage, although such information has not been provided yet. In this work, the spontaneous peptide bond cleavage was compared between Asp isomers, by applying real-time solution-state NMR to eye lens αΑ-crystallin 51-60 fragment, S51LFRTVLD58SG60 and αΒ-crystallin 61-67 analog, F61D62TGLSG67 consisting of L-α- and D-β-Asp 58 and 62, respectively. Kinetic analysis showed how tough the uncommon D-β-Asp residue was against the peptide bond cleavage as compared to natural L-α-Asp. Differences in pKa and conformation between L-α- and D-β-Asp side chains were plausible factors to determine reactivity of Asp isomers. The present study, for the first time, provides a rationale to explain less reactivity of D-β-Asp to allow abnormal accumulation.

  19. Free Energy Diagram for the Heterogeneous Enzymatic Hydrolysis of Glycosidic Bonds in Cellulose*

    PubMed Central

    Sørensen, Trine Holst; Cruys-Bagger, Nicolaj; Borch, Kim; Westh, Peter

    2015-01-01

    Kinetic and thermodynamic data have been analyzed according to transition state theory and a simplified reaction scheme for the enzymatic hydrolysis of insoluble cellulose. For the cellobiohydrolase Cel7A from Hypocrea jecorina (Trichoderma reesei), we were able to measure or collect relevant values for all stable and activated complexes defined by the reaction scheme and hence propose a free energy diagram for the full heterogeneous process. For other Cel7A enzymes, including variants with and without carbohydrate binding module (CBM), we obtained activation parameters for the association and dissociation of the enzyme-substrate complex. The results showed that the kinetics of enzyme-substrate association (i.e. formation of the Michaelis complex) was almost entirely entropy-controlled and that the activation entropy corresponded approximately to the loss of translational and rotational degrees of freedom of the dissolved enzyme. This implied that the transition state occurred early in the path where the enzyme has lost these degrees of freedom but not yet established extensive contact interactions in the binding tunnel. For dissociation, a similar analysis suggested that the transition state was late in the path where most enzyme-substrate contacts were broken. Activation enthalpies revealed that the rate of dissociation was far more temperature-sensitive than the rates of both association and the inner catalytic cycle. Comparisons of one- and two-domain variants showed that the CBM had no influence on the transition state for association but increased the free energy barrier for dissociation. Hence, the CBM appeared to promote the stability of the complex by delaying dissociation rather than accelerating association. PMID:26183776

  20. Oxytocin, the peptide that bonds the sexes also divides them

    PubMed Central

    Gao, Shan; Becker, Benjamin; Luo, Lizhu; Geng, Yayuan; Zhao, Weihua; Yin, Yu; Hu, Jiehui; Gao, Zhao; Gong, Qiyong; Hurlemann, Rene; Yao, Dezhong; Kendrick, Keith M.

    2016-01-01

    Facilitation of social attraction and bonding by the evolutionarily conserved neuropeptide oxytocin is well-established in female mammals. However, accumulating behavioral evidence suggests that oxytocin may have evolved sex-specific functional roles in the domain of human social cognition. A critical question is how oxytocin differentially modulates neural processing of social information in men and women, leading to divergent behavioral responses. Here we show that intranasal oxytocin treatment produces sex- and valence-dependent increases in amygdala activation when women view individuals identified as praising others but in men those who criticize them. Women subsequently show increased liking for the faces of these individuals, whereas in men it is reduced. Thus, oxytocin may act differentially via the amygdala to enhance the salience of positive social attributes in women but negative ones in men. We hypothesize that oxytocin may have evolved different but complementary roles to help ensure successful reproduction by encouraging mothers to promote a prosocial rearing environment for offspring and fathers to protect against antisocial influences. PMID:27325780

  1. Oxytocin, the peptide that bonds the sexes also divides them.

    PubMed

    Gao, Shan; Becker, Benjamin; Luo, Lizhu; Geng, Yayuan; Zhao, Weihua; Yin, Yu; Hu, Jiehui; Gao, Zhao; Gong, Qiyong; Hurlemann, Rene; Yao, Dezhong; Kendrick, Keith M

    2016-07-01

    Facilitation of social attraction and bonding by the evolutionarily conserved neuropeptide oxytocin is well-established in female mammals. However, accumulating behavioral evidence suggests that oxytocin may have evolved sex-specific functional roles in the domain of human social cognition. A critical question is how oxytocin differentially modulates neural processing of social information in men and women, leading to divergent behavioral responses. Here we show that intranasal oxytocin treatment produces sex- and valence-dependent increases in amygdala activation when women view individuals identified as praising others but in men those who criticize them. Women subsequently show increased liking for the faces of these individuals, whereas in men it is reduced. Thus, oxytocin may act differentially via the amygdala to enhance the salience of positive social attributes in women but negative ones in men. We hypothesize that oxytocin may have evolved different but complementary roles to help ensure successful reproduction by encouraging mothers to promote a prosocial rearing environment for offspring and fathers to protect against antisocial influences. PMID:27325780

  2. Catalytic Properties of Intramembrane Aspartyl Protease Substrate Hydrolysis Evaluated Using a FRET Peptide Cleavage Assay.

    PubMed

    Naing, Swe-Htet; Vukoti, Krishna M; Drury, Jason E; Johnson, Jennifer L; Kalyoncu, Sibel; Hill, Shannon E; Torres, Matthew P; Lieberman, Raquel L

    2015-09-18

    Chemical details of intramembrane proteolysis remain elusive despite its prevalence throughout biology. We developed a FRET peptide assay for the intramembrane aspartyl protease (IAP) from Methanoculleus marisnigri JR1 in combination with quantitative mass spectrometry cleavage site analysis. IAP can hydrolyze the angiotensinogen sequence, a substrate for the soluble aspartyl protease renin, at a predominant cut site, His-Thr. Turnover is slow (min(-1) × 10(-3)), affinity and Michaelis constant (Km) values are in the low micromolar range, and both catalytic rates and cleavage sites are the same in detergent as reconstituted into bicelles. Three well-established, IAP-directed inhibitors were directly confirmed as competitive, albeit with modest inhibitor constant (Ki) values. Partial deletion of the first transmembrane helix results in a biophysically similar but less active enzyme than full-length IAP, indicating a catalytic role. Our study demonstrates previously unappreciated similarities with soluble aspartyl proteases, provides new biochemical features of IAP and inhibitors, and offers tools to study other intramembrane protease family members in molecular detail. PMID:26118406

  3. Preparation and analysis of peptide fragments produced by pepsin hydrolysis of human plasma albumin and their relationship to its structure

    PubMed Central

    Franglen, G.; Swaniker, G. R. E.

    1968-01-01

    Human plasma albumin was prepared and subjected to proteolysis by pepsin at pH2·45 at 25° for 10min. with albumin/pepsin ratio 3000:1. Five peptide fragments were detected in the proteolysate by means of zone electrophoresis and gel filtration; these were separated and purified. Molecular weights, amino acid composition and disulphide bond content of the purified fragments were determined. The results show that a high proportion of the polypeptide chain of albumin appears to have a low cystine content, and at low pH values the molecule would be expected to have a considerable degree of freedom in its structure in these regions of the chain. A tripartite model for the structure of plasma albumin is proposed. PMID:4876098

  4. Catalysis of peptide bond formation by histidyl-histidine in a fluctuating clay environment

    NASA Technical Reports Server (NTRS)

    White, D. H.; Erickson, J. C.

    1980-01-01

    The condensation of glycine to form oligoglycines during wet-dry fluctuations on clay surfaces was enhanced up to threefold or greater by small amounts of histidyl-histidine. In addition, higher relative yields of the longer oligomers were produced. Other specific dipeptides tested gave no enhancement, and imidazole, histidine, and N-acetylhistidine gave only slight enhancements. Histidyl-histidine apparently acts as a true catalyst (in the sense of repeatedly catalyzing the reaction), since up to 52 nmol of additional glycine were incorporated into oligoglycine for each nmol of catalyst added. This is the first known instance of a peptide or similar molecule demonstrating a catalytic turnover number greater than unity in a prebiotic oligomer synthesis reaction, and suggests that histidyl-histidine is a model for a primitive prebiotic proto-enzyme. Catalysis of peptide bond synthesis by a molecule which is itself a peptide implies that related systems may be capable of exhibiting autocatalytic growth.

  5. Possible involvement of Escherichia coli 23S ribosomal RNA in peptide bond formation.

    PubMed Central

    Nitta, I; Ueda, T; Watanabe, K

    1998-01-01

    Experimental results are presented suggesting that 23S rRNA is directly involved in the peptide bond formation usually performed on the ribosome. Although several reports have indicated that the eubacterial peptidyltransferase reaction does not necessarily require all the ribosomal proteins, the reconstitution of peptidyltransferase activity by a naked 23S rRNA without the help of any of the ribosomal proteins has not been reported previously. It is demonstrated that an E. coli 23S rRNA transcript synthesized by T7 RNA polymerase in vitro was able to promote peptide bond formation in the presence of 0.5% SDS. The reaction was inhibited by the peptidyltransferase-specific antibiotics chloramphenicol and carbomycin, and by digestion with RNases A and T1. Site-directed mutageneses at two highly conserved regions close to the peptidyltransferase center ring, G2252 to U2252 and C2507G2581 to U2507A2581, also suppressed peptide bond formation. These findings strongly suggest that 23S rRNA is the peptidyltransferase itself. PMID:9510328

  6. Comparison of insect kinin analogs with cis-peptide bond motif 4-aminopyroglutamate identifies optimal stereochemistry for diuretic activity.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The insect kinins are present in a wide variety of insects and function as potent diuretic peptides, though they are subject to rapid degradation by internal peptidases. Insect kinin analogs incorporating stereochemical variants of (2S,4S)-4-aminopyroglutamate (APy), a cis-peptide bond motif, demon...

  7. Characterization of Sviceucin from Streptomyces Provides Insight into Enzyme Exchangeability and Disulfide Bond Formation in Lasso Peptides.

    PubMed

    Li, Yanyan; Ducasse, Rémi; Zirah, Séverine; Blond, Alain; Goulard, Christophe; Lescop, Ewen; Giraud, Caroline; Hartke, Axel; Guittet, Eric; Pernodet, Jean-Luc; Rebuffat, Sylvie

    2015-11-20

    Lasso peptides are bacterial ribosomally synthesized and post-translationally modified peptides. They have sparked increasing interest in peptide-based drug development because of their compact, interlocked structure, which offers superior stability and protein-binding capacity. Disulfide bond-containing lasso peptides are rare and exhibit highly sought-after activities. In an effort to expand the repertoire of such molecules, we heterologously expressed, in Streptomyces coelicolor, the gene cluster encoding sviceucin, a type I lasso peptide with two disulfide bridges originating from Streptomyces sviceus, which allowed it to be fully characterized. Sviceucin and its reduced forms were characterized by mass spectrometry and peptidase digestion. The three-dimensional structure of sviceucin was determined using NMR. Sviceucin displayed antimicrobial activity selectively against Gram-positive bacteria and inhibition of fsr quorum sensing in Enterococcus faecalis. This study adds sviceucin to the type I lasso peptide family as a new representative. Moreover, new clusters encoding disulfide-bond containing lasso peptides from Actinobacteria were identified by genome mining. Genetic and functional analyses revealed that the formation of disulfide bonds in sviceucin does not require a pathway-encoded thiol-disulfide oxidoreductase. Most importantly, we demonstrated the functional exchangeability of the sviceucin and microcin J25 (a non-disulfide-bridged lasso peptide) macrolactam synthetases in vitro, highlighting the potential of hybrid lasso synthetases in lasso peptide engineering. PMID:26343290

  8. Mass Spectrometry Analysis of the Extracellular Peptidome of Lactococcus lactis: Lines of Evidence for the Coexistence of Extracellular Protein Hydrolysis and Intracellular Peptide Excretion.

    PubMed

    Guillot, Alain; Boulay, Mylène; Chambellon, Émilie; Gitton, Christophe; Monnet, Véronique; Juillard, Vincent

    2016-09-01

    We report here the use of a peptidomic approach to revisit the extracellular proteolysis of Lactococcus lactis. More than 1800 distinct peptides accumulate externally during growth of the plasmid-free protease-negative strain L. lactis IL1403 in a protein- and peptide-free medium. These peptides mainly originate from cell-surface- and cytoplasmic-located proteins, despite the fact that no cell lysis could be evidenced. Positioning each identified peptide on its parental protein sequence demonstrated the involvement of exo- and endopeptidase activities. The endopeptidases responsible for the release of surface and cytoplasmic peptides had distinct specificities. The membrane-anchored protease HtrA was responsible for the release of only a part of the surface peptides, and its preference for branched-chain amino acids in the N-terminal side of the cleaved bond was established in situ. Other yet uncharacterized surface proteases were also involved. Several lines of evidence suggest that surface and cytoplasmic peptides were produced by different routes, at least part of the latter being most likely excreted as peptides from the cells. The mechanism by which these cytoplasmic peptides are excreted remains an open question, as it is still the case for excreted cytoplasmic proteins. PMID:27439475

  9. Mass spectral study of hybrid peptides derived from (R)-aminoxy ester and [beta]-amino acids: The influence of aminoxy peptide bond (CO-NH-O) on peptide fragmentation under electrospray ionization conditions

    NASA Astrophysics Data System (ADS)

    Ramesh, V.; Ramesh, M.; Srinivas, R.; Sharma, G. V. M.; Manohar, V.

    2009-04-01

    A new class of Boc-protected aminoxy hybrid peptides containing repeats of [beta]-hAla-(R)-Ama-, and [beta]-Caa-(R)-Ama- ([beta]-hAla = [beta]3-(S)-hAlanine, (R)-Ama = (R)-aminoxy ester, and [beta]-Caa = (R)-C-linked carbo-[beta]3-amino acid) have been studied by electrospray ionization (ESI) ion-trap and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) of their protonated, cationized, and negative ions. MS3 CID of protonated aminoxy peptides of [beta]-hAla-(R)-Ama- yield intense [beta]-amino acid characteristic retro-Mannich fragmentation. The bn+ and [bn-methyl imine]+ (n = 3, 5) ions formed by cleavage of aminoxy peptide bond (CO-NH-O) are more intense than bn+ (n = 2, 4) formed by that of peptide bond (CO-NH-C) cleavage. Another characteristic ion observed is due to loss of H3NO from yn+ ions. The cationized (Li+, and Na+) peptides dissociate differently compared to protonated peptides. Intense cationized cn and zn ions are formed due to the cleavage of N-O bond. The deprotonated peptides also show abundant cn- and zn- ions (n = 1, 3, 5) and do not form any yn- ions. All these results clearly indicate the influence of aminoxy peptide bond on fragmentation of these hybrid peptides.

  10. Novel Cβ-Cγ Bond Cleavages of Tryptophan-Containing Peptide Radical Cations

    NASA Astrophysics Data System (ADS)

    Song, Tao; Hao, Qiang; Law, Chun-Hin; Siu, Chi-Kit; Chu, Ivan K.

    2012-02-01

    In this study, we observed unprecedented cleavages of the Cβ-Cγ bonds of tryptophan residue side chains in a series of hydrogen-deficient tryptophan-containing peptide radical cations (M•+) during low-energy collision-induced dissociation (CID). We used CID experiments and theoretical density functional theory (DFT) calculations to study the mechanism of this bond cleavage, which forms [M - 116]+ ions. The formation of an α-carbon radical intermediate at the tryptophan residue for the subsequent Cβ-Cγ bond cleavage is analogous to that occurring at leucine residues, producing the same product ions; this hypothesis was supported by the identical product ion spectra of [LGGGH - 43]+ and [WGGGH - 116]+, obtained from the CID of [LGGGH]•+ and [WGGGH]•+, respectively. Elimination of the neutral 116-Da radical requires inevitable dehydrogenation of the indole nitrogen atom, leaving the radical centered formally on the indole nitrogen atom ([Ind]•-2), in agreement with the CID data for [WGGGH]•+ and [W1-CH3GGGH]•+; replacing the tryptophan residue with a 1-methyltryptophan residue results in a change of the base peak from that arising from a neutral radical loss (116 Da) to that arising from a molecule loss (131 Da), both originating from Cβ-Cγ bond cleavage. Hydrogen atom transfer or proton transfer to the γ-carbon atom of the tryptophan residue weakens the Cβ-Cγ bond and, therefore, decreases the dissociation energy barrier dramatically.

  11. Enhancing Peptide Ligand Binding to Vascular Endothelial Growth Factor by Covalent Bond Formation

    PubMed Central

    Marquez, Bernadette V.; Beck, Heather E.; Aweda, Tolulope A.; Phinney, Brett; Holsclaw, Cynthia; Jewell, William; Tran, Diana; Day, Jeffrey J.; Peiris, Malalage N.; Nwosu, Charles; Lebrilla, Carlito; Meares, Claude F.

    2012-01-01

    Formation of a stable covalent bond between a synthetic probe molecule and a specific site on a target protein has many potential applications in biomedical science. For example, the properties of probes used as receptor-imaging ligands may be improved by increasing their residence time on the targeted receptor. Among the more interesting cases are peptide ligands, the strongest of which typically bind to receptors with micromolar dissociation constants, and which may depend on processes other than simple binding to provide images. The side chains of cysteine, histidine, or lysine are attractive for chemical attachment to improve binding to a receptor protein, and a system based on acryloyl probes attaching to engineered cysteine provides excellent positron emission tomographic images in animal models (Wei et al. (2008) J. Nucl. Med. 49, 1828-1835). In nature, lysine is a more common but less reactive residue than cysteine, making it an interesting challenge to modify. To seek practically useful cross-linking yields with naturally occurring lysine side chains, we have explored not only acryloyl but also other reactive linkers with different chemical properties. We employed a peptide-VEGF model system to discover that a 19mer peptide ligand, which carried a lysine-tagged dinitrofluorobenzene group, became attached stably and with good yield to a unique lysine residue on human vascular endothelial growth factor (VEGF), even in the presence of 70% fetal bovine serum. The same peptide carrying acryloyl and related Michael acceptors gave low yields of attachment to VEGF, as did the chloroacetyl peptide. PMID:22537066

  12. Magnesium ion catalyzed P-N bond hydrolysis in imidazolide-activated nucleotides - Relevance to template-directed synthesis of polynucleotides

    NASA Technical Reports Server (NTRS)

    Kanavarioti, Anastassia; Bernasconi, Claude F.; Doodokyan, Donald L.; Alberas, Diann J.

    1989-01-01

    Results are presented from a detailed study of the P-N bond hydrolysis in guanosine 5-prime-monophosphate 2-methylimidazolide (2-MeImpG) and in guanosine 5-prime-imidazolide (ImpG) in the presence of 0-0.50 M Mg(2+). Pseudo-first-order rate constants of these compounds were obtained as a function of Mg(2+) concentration, for pH values between 6 and 10 and 37 C. It was found that Mg(2+) catalysis was most effective at pH 10, where a 15-fold increase in hydrolysis was achieved in 0.02 M Mg; at 0.2 M, a 115-fold increase was observed. Implication of these results for the mechanism of template-directed oligomerization is discussed.

  13. Multinuclear diffusion NMR spectroscopy and DFT modeling: a powerful combination for unraveling the mechanism of phosphoester bond hydrolysis catalyzed by metal-substituted polyoxometalates.

    PubMed

    Luong, Thi Kim Nga; Shestakova, Pavletta; Mihaylov, Tzvetan T; Absillis, Gregory; Pierloot, Kristine; Parac-Vogt, Tatjana N

    2015-03-01

    A detailed reaction mechanism is proposed for the hydrolysis of the phosphoester bonds in the DNA model substrate bis(4-nitrophenyl) phosphate (BNPP) in the presence of the Zr(IV)-substituted Keggin type polyoxometalate (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]⋅7 H2O (ZrK 2:2) at pD 6.4. Low-temperature (31)P DOSY spectra at pD 6.4 gave the first experimental evidence for the presence of ZrK 1:1 in fast equilibrium with ZrK 2:2 in purely aqueous solution. Moreover, theoretical calculations identified the ZrK 1:1 form as the potentially active species in solution. The reaction intermediates involved in the hydrolysis were identified by means of (1)H/(31)P NMR studies, including EXSY and DOSY NMR spectroscopy, which were supported by DFT calculations. This experimental/theoretical approach enabled the determination of the structures of four intermediate species in which the starting compound BNPP, nitrophenyl phosphate (NPP), or the end product phosphate (P) is coordinated to ZrK 1:1. In the proposed reaction mechanism, BNPP initially coordinates to ZrK 1:1 in a monodentate fashion, which results in hydrolysis of the first phosphoester bond in BNPP and formation of NPP. EXSY NMR studies showed that the bidentate complex between NPP and ZrK 1:1 is in equilibrium with monobound and free NPP. Subsequently, hydrolysis of NPP results in P, which is in equilibrium with its monobound form. PMID:25652658

  14. Rapid sequencing and disulfide mapping of peptides containing disulfide bonds by using 1,5-diaminonaphthalene as a reductive matrix.

    PubMed

    Fukuyama, Yuko; Iwamoto, Shinichi; Tanaka, Koichi

    2006-02-01

    MS/MS is indispensable for the amino acid sequencing of peptides. However, its use is limited for peptides containing disulfide bonds. We have applied the reducing properties of 1,5-diaminonaphthalene (1,5-DAN) as a MALDI matrix to amino acid sequencing and disulfide bond mapping of human urotensin II possessing one disulfide bond, and human guanylin possessing two disulfide bonds. 1,5-DAN was used in the same manner as the usual MALDI matrices without any pre-treatment of the peptide, and MS/MS was performed using a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer (MALDI QIT TOFMS). The results demonstrated that MS/MS of the molecular ions reduced by 1,5-DAN provided a series of significant b-/y-product ions. All 11 amino acid residues of urotensin II were identified using 1,5-DAN, while only 5 out of 11 residues were identified using 2,5-dihydroxybenzoic acid (DHB); similarly 11 out of 15 amino acid residues of guanylin were identified using 1,5-DAN, while only three were identified using DHB. In addition, comparison of the theoretical and measured values of the mass differences between corresponding MS/MS product ions using 1,5-DAN and DHB narrowed down the possible disulfide bond arrangement candidates. Consequently, 1,5-DAN as a reductive matrix facilitates rapid amino acid sequencing and disulfide mapping for peptides containing disulfide bonds. PMID:16382486

  15. Novel Cβ-Cγ bond cleavages of tryptophan-containing peptide radical cations.

    PubMed

    Song, Tao; Hao, Qiang; Law, Chun-Hin; Siu, Chi-Kit; Chu, Ivan K

    2012-02-01

    In this study, we observed unprecedented cleavages of the C(β)-C(γ) bonds of tryptophan residue side chains in a series of hydrogen-deficient tryptophan-containing peptide radical cations (M(•+)) during low-energy collision-induced dissociation (CID). We used CID experiments and theoretical density functional theory (DFT) calculations to study the mechanism of this bond cleavage, which forms [M - 116](+) ions. The formation of an α-carbon radical intermediate at the tryptophan residue for the subsequent C(β)-C(γ) bond cleavage is analogous to that occurring at leucine residues, producing the same product ions; this hypothesis was supported by the identical product ion spectra of [LGGGH - 43](+) and [WGGGH - 116](+), obtained from the CID of [LGGGH](•+) and [WGGGH](•+), respectively. Elimination of the neutral 116-Da radical requires inevitable dehydrogenation of the indole nitrogen atom, leaving the radical centered formally on the indole nitrogen atom ([Ind](•)-2), in agreement with the CID data for [WGGGH](•+) and [W(1-CH3)GGGH](•+); replacing the tryptophan residue with a 1-methyltryptophan residue results in a change of the base peak from that arising from a neutral radical loss (116 Da) to that arising from a molecule loss (131 Da), both originating from C(β)-C(γ) bond cleavage. Hydrogen atom transfer or proton transfer to the γ-carbon atom of the tryptophan residue weakens the C(β)-C(γ) bond and, therefore, decreases the dissociation energy barrier dramatically. PMID:22135037

  16. Intermolecular charge flux as the origin of infrared intensity enhancement upon halogen-bond formation of the peptide group

    NASA Astrophysics Data System (ADS)

    Torii, Hajime

    2010-07-01

    The changes in the vibrational properties of the peptide group upon formation of O⋯X and N⋯X halogen bonds are studied theoretically. Calculations are carried out for complexes of N-methylacetamide (NMA), a well known model molecule of the peptide group, with halogen-containing molecules. For comparison, calculations are also carried out for some NMA-water hydrogen-bonding complexes. It is shown that the infrared (IR) intensity of the amide I mode of the peptide group is enhanced significantly (up to about 520 km mol-1 or 2.6 times) upon CO⋯X halogen-bond formation, in spite of rather modest magnitudes of the intermolecular electric field and of the changes in the CO bond length and in the amide I vibrational frequency as compared with the cases of the CO⋯H(D) hydrogen bonding. From the analysis of the changes in the dipole derivative and in the electronic structure, it is shown that this IR intensity enhancement arises from the intermolecular charge flux. For the N⋯X halogen bonding complexes, some characteristic changes in the vibrational properties are seen, among which the IR intensity enhancement of the ND out-of-plane wagging mode is most notable. The reason why such large IR intensity enhancements are seen for these particular vibrational modes is examined.

  17. The effect of smectite composition on the catalysis of peptide bond formation.

    PubMed

    Bujdák, J; Rode, B M

    1996-10-01

    Clay-catalyzed glycine and diglycine oligomerizations were performed as drying/wetting cycles at 80 degrees C. Two trioctahedral smectites (hectorite and saponite), three pure montmorillonites, a ferruginous smectite, an Fe(II)-rich smectite, and three smectites containing goethite admixture were used as catalysts. Highest peptide bond formation was found with trioctahedral smectites. About 7% of glycine was converted to diglycine and diketopiperazine on hectorite after 7 days. In the case of dioctahedral smectites, highest yields were achieved using clays with a negative-layer charge localized in the octahedral sheets (up to 2% of converted glycine after 7 days). The presence of Fe(II) in clay is reflected in a higher efficiency in catalyzing amino acid dimerization (about 3.5% of converted glycine after 7 days). The possible significance of the results for prebiotic chemistry is discussed. PMID:8798338

  18. Effects of tea saponin on glucan conversion and bonding behaviour of cellulolytic enzymes during enzymatic hydrolysis of corncob residue with high lignin content

    PubMed Central

    2013-01-01

    Background Recently, interest in the utilization of corncob residue (CCR, with high lignin of 45.1%) as a feedstock for bioethanol has been growing. Surfactants have been one of the most popular additives intended to prevent the inhibitory effect of lignin on cellulolytic enzymes, thereby improving hydrolysis. In this study, the effects of biosurfactant tea saponin (TS) on the enzymatic hydrolysis of CCR and the bonding behavior of cellulolytic enzymes to the substrate were investigated. The surface tension in the supernatant was also detected to obtain information about the characteristics and stability of TS. Results The glucose concentration was 17.15 mg/mL at 120 hours of hydrolysis with the low loading of cellulolytic enzymes (7.0 FPU/g cellulose and 10.5 BGU/g cellulose) and 5% CCR. The optimal dosage of TS was its critical micelle concentration (cmc, 1.80 mg/mL). The glucose yield was enhanced from 34.29 to 46.28 g/100 g dry matter by TS. The results indicate that TS can promote the adsorption of cellulolytic enzymes on the substrate and mediate the release of adsorbed enzymes. Meanwhile, TS improves the recovery of the cellulolytic enzymes after a hydrolysis cycle and prevents deactivation of the enzymes during the intense shaking process. The surface tension in supernatants of digested CCR with TS remained at 50.00 mN/m during the course of hydrolysis. It is interesting to note that biosurfactant TS can maintain the surface tension in supernatants, despite its digestibility by cellulolytic enzymes. Conclusions Serving as an accelerant of lignocellulose hydrolysis, TS can also be degraded by the cellulolytic enzymes and release glucose while retaining stability, which reduces the cost of both the cellulolytic enzymes and the additive. As the glucose from the TS could be utilized by yeast, further efforts will investigate the mechanism of function and the application of TS in the production of ethanol by simultaneous saccharification and fermentation

  19. Phi ({Phi}) and psi ({Psi}) angles involved in malarial peptide bonds determine sterile protective immunity

    SciTech Connect

    Patarroyo, Manuel E.; Moreno-Vranich, Armando; Bermudez, Adriana

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer Phi ({Phi}) and psi ({Psi}) angles determine sterile protective immunity. Black-Right-Pointing-Pointer Modified peptide's tendency to assume a regular conformation related to a PPII{sub L}. Black-Right-Pointing-Pointer Structural modifications in mHABPs induce Ab and protective immunity. Black-Right-Pointing-Pointer mHABP backbone atom's interaction with HLA-DR{beta}1{sup Asterisk-Operator} is stabilised by H-bonds. -- Abstract: Modified HABP (mHABP) regions interacting with HLA-DR{beta}1{sup Asterisk-Operator} molecules have a more restricted conformation and/or sequence than other mHABPs which do not fit perfectly into their peptide binding regions (PBR) and do not induce an acceptable immune response due to the critical role of their {Phi} and {Psi} torsion angles. These angle's critical role was determined in such highly immunogenic, protection-inducing response against experimental malaria using the conformers (mHABPs) obtained by {sup 1}H-NMR and superimposed into HLA-DR{beta}1{sup Asterisk-Operator }-like Aotus monkey molecules; their phi ({Phi}) and psi ({Psi}) angles were measured and the H-bond formation between these molecules was evaluated. The aforementioned mHABP propensity to assume a regular conformation similar to a left-handed polyproline type II helix (PPII{sub L}) led to suggesting that favouring these conformations according to their amino acid sequence would lead to high antibody titre production and sterile protective immunity induction against malaria, thereby adding new principles or rules for vaccine development, malaria being one of them.

  20. Metabolism of saikosaponin a in rats: diverse oxidations on the aglycone moiety in liver and intestine in addition to hydrolysis of glycosidic bonds.

    PubMed

    Liu, Guoqiang; Tian, Yuan; Li, Geng; Xu, Lei; Song, Rui; Zhang, Zunjian

    2013-03-01

    The main objective of the present study was to completely characterize the metabolites of the triterpenoid saikosaponin a (SSa) in rats. To this aim, we compared the metabolites in plasma, bile, urine, and feces samples following oral and i.v. routes of administration using liquid chromatography-diode array detector coupled with hybrid ion trap-time-of-flight mass spectrometry. As a result, besides 2 known metabolites, prosaikogenin f and saikogenin f, 15 new metabolites were detected in all. It was found that SSa is metabolized mainly in phase I manner, i.e., hydration and monooxidation on the aglycone moiety and hydrolysis of the β-glucosidic bond in the liver, and sequential hydrolysis of β-glucosidic and β-fucosidic bonds followed by dehydrogenation, hydroxylation, carboxylation, and combinations of these steps on the aglycone moiety in the intestinal tract. Both the renal and biliary routes were observed for the excretion of SSa and its metabolites. Further, a clear metabolic profile in rats was proposed in detail according to the results from the in vivo animal experiment after different routes of administration. Our results update the preclinical metabolism and disposition data on SSa, which is not only helpful for the future human metabolic study of this compound but also provides basic information for better understanding of the efficacy and safety of prescriptions containing saikosaponins. PMID:23277344

  1. PEP-FOLD: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides

    PubMed Central

    Thévenet, Pierre; Shen, Yimin; Maupetit, Julien; Guyon, Frédéric; Derreumaux, Philippe; Tufféry, Pierre

    2012-01-01

    In the context of the renewed interest of peptides as therapeutics, it is important to have an on-line resource for 3D structure prediction of peptides with well-defined structures in aqueous solution. We present an updated version of PEP-FOLD allowing the treatment of both linear and disulphide bonded cyclic peptides with 9–36 amino acids. The server makes possible to define disulphide bonds and any residue–residue proximity under the guidance of the biologists. Using a benchmark of 34 cyclic peptides with one, two and three disulphide bonds, the best PEP-FOLD models deviate by an average RMS of 2.75 Å from the full NMR structures. Using a benchmark of 37 linear peptides, PEP-FOLD locates lowest-energy conformations deviating by 3 Å RMS from the NMR rigid cores. The evolution of PEP-FOLD comes as a new on-line service to supersede the previous server. The server is available at: http://bioserv.rpbs.univ-paris-diderot.fr/PEP-FOLD. PMID:22581768

  2. Note: Charge transfer in a hydrated peptide group is determined mainly by its intrinsic hydrogen-bond energetics

    SciTech Connect

    Mirkin, Noemi G.; Krimm, Samuel

    2014-01-28

    Charge transfer in a hydrogen-bonded N-methylacetamide(H{sub 2}O){sub 3} system is obtained from ωB97X-D/6-31++G** and CHelpG atomic charge calculations of individual peptide-water interactions as well as that of the entire complex. In the latter, the electron transfer to water is 0.19 e, influenced primarily by the hydrogen bonds to the C=O group. The values of such charge transfer are paralleled by the corresponding intrinsic hydrogen-bond energies. These results support the desirability of incorporating charge transfer in molecular mechanics energy functions.

  3. Synthesized Peptides from Yam Dioscorin Hydrolysis in Silico Exhibit Dipeptidyl Peptidase-IV Inhibitory Activities and Oral Glucose Tolerance Improvements in Normal Mice.

    PubMed

    Lin, Yin-Shiou; Han, Chuan-Hsiao; Lin, Shyr-Yi; Hou, Wen-Chi

    2016-08-24

    RRDY, RL, and DPF were the top 3 of 21 peptides for inhibitions against dipeptidyl peptidase-IV (DPP-IV) from the pepsin hydrolysis of yam dioscorin in silico and were further investigated in a proof-of-concept study in normal ICR mice for regulating glucose metabolism by the oral glucose tolerance test (OGTT). The sample or sitagliptin (positive control) was orally administered by a feeding gauge; 30 min later, the glucose loads (2.5 g/kg) were performed. RRDY, yam dioscorin, or sitagliptin preload, but not DPF, lowered the area under the curve (AUC0-120) of blood glucose and DPP-IV activity and elevated the AUC0-120 of blood insulin, which showed significant differences compared to control (P < 0.05 or 0.001). These results suggested that RRDY and yam dioscorin might be beneficial in glycemic control in normal mice and need further investigations in diabetic animal models. PMID:27499387

  4. Increased yield of high purity recombinant human brain natriuretic peptide by acid hydrolysis of short fusion partner in Escherichia coli.

    PubMed

    Kanumuri, Radha Madhavi; Bajji, Chitra; Tummuru, Rajesh R; Tatireddigari, Venkat R R Arva; Mangamoori, Lakshmi Narasu; Panati, Kalpana; Narala, Venkata Ramireddy

    2015-07-01

    Recombinant human B-type natriuretic peptide (rhBNP) is a 32-amino acid peptide used to treat congestive heart failure. In this paper, we report a method for the increased production of rhBNP in Escherichia coli with high purity. hBNP was cloned with a short growth hormone fusion partner coupled with a unique acid-labile dipeptide linker to cleave the fusion protein to release the rhBNP. The recombinant fusion protein was expressed as an inclusion body (IB) and the fermentation process was optimized to produce on large scale. The IBs were recovered by cell lysis, and the pure IBs were directly treated with diluted acid to get the target peptide from the fusion protein and the resultant peptide was purified by reversed phase chromatography. The final purity of the rhBNP was more than 99% with yield of 50mg per liter of culture, which is ten times higher than the previous reports. The purified rhBNP exhibited specific biological activity similar to the standard peptide in producing cyclic-guanosine monophosphate. PMID:25823948

  5. The use of sub-critical water hydrolysis for the recovery of peptides and free amino acids from food processing wastes. Review of sources and main parameters.

    PubMed

    Marcet, Ismael; Álvarez, Carlos; Paredes, Benjamín; Díaz, Mario

    2016-03-01

    Food industry processing wastes are produced in enormous amounts every year, such wastes are usually disposed with the corresponding economical cost it implies, in the best scenario they can be used for pet food or composting. However new promising technologies and tools have been developed in the last years aimed at recovering valuable compounds from this type of materials. In particular, sub-critical water hydrolysis (SWH) has been revealed as an interesting way for recovering high added-value molecules, and its applications have been broadly referred in the bibliography. Special interest has been focused on recovering protein hydrolysates in form of peptides or amino acids, from both animal and vegetable wastes, by means of SWH. These recovered biomolecules have a capital importance in fields such as biotechnology research, nutraceuticals, and above all in food industry, where such products can be applied with very different objectives. Present work reviews the current state of art of using sub-critical water hydrolysis for protein recovering from food industry wastes. Key parameters as reaction time, temperature, amino acid degradation and kinetic constants have been discussed. Besides, the characteristics of the raw material and the type of products that can be obtained depending on the substrate have been reviewed. Finally, the application of these hydrolysates based on their functional properties and antioxidant activity is described. PMID:26831563

  6. Cleavage of peptide bonds bearing ionizable amino acids at P{sub 1} by serine proteases with hydrophobic S{sub 1} pocket

    SciTech Connect

    Qasim, Mohammad A.; Song, Jikui; Markley, John L.; Laskowski, Michael

    2010-10-01

    Research highlights: {yields} Large pK shifts in ionizable groups when buried in the protein interior. {yields} Substrate dependent shifts in pH optimum for serine proteases. {yields} Lys side chain is a stronger acid in serine protease S{sub 1} pocket than Asp side chain. -- Abstract: Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx = Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3-11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5-8.0, 5.5-6.0, and {approx}10, respectively. At the normally reported pH optimum (pH 7-8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The pK values of the side chains of Asp and Lys in the hydrophobic S{sub 1} pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.

  7. IRMPD Spectroscopy: Evidence of Hydrogen Bonding in the Gas Phase Conformations of Lasso Peptides and their Branched-Cyclic Topoisomers.

    PubMed

    Jeanne Dit Fouque, Kevin; Lavanant, Hélène; Zirah, Séverine; Steinmetz, Vincent; Rebuffat, Sylvie; Maître, Philippe; Afonso, Carlos

    2016-06-01

    Lasso peptides are natural products characterized by a mechanically interlocked topology. The conformation of lasso peptides has been probed in the gas phase using ion mobility-mass spectrometry (IM-MS) which showed differences in the lasso and their unthreaded branched-cyclic topoisomers depending on the ion charge states. To further characterize the evolution of gas phase conformations as a function of the charge state and to assess associated changes in the hydrogen bond network, infrared multiple photon dissociation (IRMPD) action spectroscopy was carried out on two representative lasso peptides, microcin J25 (MccJ25) and capistruin, and their branched-cyclic topoisomers. For the branched-cyclic topoisomers, spectroscopic evidence of a disruption of neutral hydrogen bonds were found when comparing the 3+ and 4+ charge states. In contrast, for the lasso peptides, the IRMPD spectra were found to be similar for the two charge states, suggesting very little difference in gas phase conformations upon addition of a proton. The IRMPD data were thus found consistent and complementary to IM-MS, confirming the stable and compact structure of lasso peptides in the gas phase. PMID:27171649

  8. Conformational stability of alpha-lactalbumin missing a peptide bond between Asp66 and Pro67.

    PubMed

    Hamada, S; Moriyama, Y; Yamaguchi, K; Takeda, K

    1994-05-01

    The peptide bond between Asp66-Pro67 of alpha-lactalbumin was cleaved with formic acid (cleaved alpha-lactalbumin). Secondary structural changes of the cleaved alpha-lactalbumin, in which the two separated polypeptides were joined by disulfide bridges, were examined in solutions of sodium dodecyl sulfate (SDS), urea, and guanidine hydrochloride. The structural changes of the cleaved alpha-lactalbumin were compared with those of the intact protein. The relative proportions of secondary structures were determined by curve fitting of the circular dichroism. The cleaved alpha-lactalbumin contained 29% alpha-helical structure as against 34% for the intact protein. Some helices of the cleaved alpha-lactalbumin which had been disrupted by the cleavage appeared to be reformed upon the addition of SDS of very low concentration (0.5 mM). In the SDS solution, the helicities of both the intact and cleaved proteins increased, attaining 44% at 4 mM SDS. On the other hand, the helical structures of the cleaved alpha-lactalbumin began to be disrupted at low concentrations of guanidine hydrochloride and urea compared with that of the intact protein. However, no difference was observed in the thermal denaturations of the intact and cleaved proteins, except for the difference in the original helicities. The helicities of both proteins decreased with an increase of temperature up to 65 degrees C and recovered upon cooling. PMID:7986345

  9. Counting sulfhydryls and disulfide bonds in peptides and proteins using mercurial ions as an MS-tag.

    PubMed

    Guo, Yifei; Chen, Liqin; Yang, Limin; Wang, Qiuquan

    2008-08-01

    Organic mercurial compounds are the most specific and sensitive reagents for reaction with the sulfhydryl groups (SHs) in peptides and proteins because of the strong mercury-sulfur affinity. Using the monofunctional organic mercury ion RHg(+) as a mass spectrometry (MS)-tag has the advantages of reacting with one sulfhydryl group, offering definite mass shift, and especially stable and characteristic nonradioactive isotopic distribution. Mass spectrometric analysis of derivatized sulfhydryls in peptides/proteins is thus an alternative for precisely counting the number of sulfhydryl groups and disulfide bonds (SS). Here the tags used include monomethylmercury chloride, monoethylmercury chloride, and 4-(hydroxymercuri) benzoic acid. The feasibility of this strategy is demonstrated using HPLC/ESI-MS to count SHs and SS in model peptides/proteins, i.e., glutathione, phytochelatins, lysozyme and beta-lactoglobulin, which contain increasing SHs and various SS linkages. PMID:18524619

  10. Site-Specific Conjugation of Peptides and Proteins via Rebridging of Disulfide Bonds Using the Thiol-Yne Coupling Reaction.

    PubMed

    Griebenow, Nils; Dilmaç, Alicia M; Greven, Simone; Bräse, Stefan

    2016-04-20

    Herein, we describe an extension of our previously reported photomediated disulfide rebridging methodology to the conjugation of peptides and proteins. The methodology proved to be reproducible with various alkynes and different peptides. This study includes the first rebridging of the disulfide bond of a peptide through a thiol-yne reaction with a cyclooctyne. In all cases, the rebridging was proven by MS analyses and confirmed by the absence of olefinic protons on (1)H NMR spectra of the resulting products. Finally, this one-pot reduction thiol-yne conjugation was successfully applied to an antibody Fab fragment with a promising conversion, which set a good ground for the future syntheses of new protein and antibody conjugates. PMID:27031217

  11. An activated triple bond linker enables ‘click’ attachment of peptides to oligonucleotides on solid support

    PubMed Central

    Wenska, Malgorzata; Alvira, Margarita; Steunenberg, Peter; Stenberg, Åsa; Murtola, Merita; Strömberg, Roger

    2011-01-01

    A general procedure, based on a new activated alkyne linker, for the preparation of peptide–oligonucleotide conjugates (POCs) on solid support has been developed. With this linker, conjugation is effective at room temperature (RT) in millimolar concentration and submicromolar amounts. This is made possible since the use of a readily attachable activated triple bond linker enhances the Cu(I) catalyzed 1,3-dipolar cycloaddition (‘click’ reaction). The preferred scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of (i) an H-phosphonate-based aminolinker; (ii) the triple bond donor p-(N-propynoylamino)toluic acid (PATA); and (iii) azido-functionalized peptides. The method gives conversion of oligonucleotide to the POC on solid support, and only involves a single purification step after complete assembly. The synthesis is flexible and can be carried out without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. Methodology for the ready conversion of peptides into ‘clickable’ azidopeptides with the possibility of selecting either N-terminus or C-terminus connection also adds to the flexibility and usability of the method. Examples of synthesis of POCs include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals. PMID:21795380

  12. Immobilization of collagen peptide on dialdehyde bacterial cellulose nanofibers via covalent bonds for tissue engineering and regeneration

    PubMed Central

    Wen, Xiaoxiao; Zheng, Yudong; Wu, Jian; Wang, Lu-Ning; Yuan, Zhenya; Peng, Jiang; Meng, Haoye

    2015-01-01

    Bacterial cellulose (BC) is an alternative nanostructured biomaterial to be utilized for a wide range of biomedical applications. Because of its low bioactivity, which restricted its practical application, collagen and collagen hydrolysate were usually composited into BC. It is necessary to develop a new method to generate covalent bonds between collagen and cellulose to improve the immobilization of collagen on BC. This study describes a facile dialdehyde BC/collagen peptide nanocomposite. BC was oxidized into dialdehyde bacterial cellulose (DBC) by regioselective oxidation, and then composited with collagen peptide (Col-p) via covalent bonds to form Schiff’s base type compounds, which was demonstrated by the results of microstructures, contact angle, Col-p content, and peptide-binding ratio. The peptide-binding ratio was further affected by the degree of oxidation, pH value, and zeta potential. In vitro desorption measurement of Col-p suggested a controlled release mechanism of the nanocomposite. Cell tests indicated that the prepared DBC/Col-p composite was bioactive and suitable for cell adhesion and attachment. This work demonstrates that the DBC/Col-p composite is a promising material for tissue engineering and regeneration. PMID:26229466

  13. The Oxidatively-Induced DNA Lesions 8,5′-Cyclo-2′-Deoxyadenosine and 8-Hydroxy-2′-Deoxyadenosine Are Strongly Resistant to Acid-Induced Hydrolysis of the Glycosidic Bond

    PubMed Central

    Theruvathu, Jacob A.; Jaruga, Pawel; Dizdaroglu, Miral

    2007-01-01

    The 8,5’-cyclopurine-2’-deoxynucleosides (cPu) are unique oxidatively-induced DNA lesions in that they are specifically repaired by NER. In the absence of NER, a possible mechanism for cPu removal is spontaneous glycosidic bond hydrolysis followed by enzymic processing. Such a mechanism could be significant if the glycosidic bond in cPu were substantially destabilized, as shown for other DNA lesions. Therefore, we investigated the stability of the glycosidic bond in a cPu, 8,5’(S)-cyclo-dA (S-cdA) against acid hydrolysis. For comparison, we also studied 8-OH-dA. We found that the glycosidic bond in S-cdA is ≈40 fold more resistant to glycosidic bond hydrolysis compared to dA. Interestingly, under the same conditions, the glycosidic bond in 8-OH-dA was even more stable than in S-cdA. These studies effectively rule out any mechanism for the removal of S-cdA or 8-OH-dA from DNA that requires spontaneous glycosidic bond hydrolysis, and further support the proposed role of cPu in the neurodegeneration observed in xeroderma pigmentosum patients who lack NER. Of broader significance, since NER does not function in non-transcribed DNA sequences of terminally differentiated cells, including neurons, cPu are expected to accumulate in such sequences even in individuals with normal NER, which could be important in the ageing process. PMID:17692895

  14. Role of the P-F bond in fluoride-promoted aqueous VX hydrolysis: an experimental and theoretical study.

    PubMed

    Marciano, Daniele; Columbus, Ishay; Elias, Shlomi; Goldvaser, Michael; Shoshanim, Ofir; Ashkenazi, Nissan; Zafrani, Yossi

    2012-11-16

    Following our ongoing studies on the reactivity of the fluoride ion toward organophosphorus compounds, we established that the extremely toxic and environmentally persistent chemical warfare agent VX (O-ethyl S-2-(diisopropylamino)ethyl methylphosphonothioate) is exclusively and rapidly degraded to the nontoxic product EMPA (ethyl methylphosphonic acid) even in dilute aqueous solutions of fluoride. The unique role of the P-F bond formation in the reaction mechanism was explored using both experimental and computational mechanistic studies. In most cases, the "G-analogue" (O-ethyl methylphosphonofluoridate, Et-G) was observed as an intermediate. Noteworthy and of practical importance is the fact that the toxic side product desethyl-VX, which is formed in substantial quantities during the slow degradation of VX in unbuffered water, is completely avoided in the presence of fluoride. A computational study on a VX-model, O,S-diethyl methylphosphonothioate (1), clarifies the distinctive tendency of aqueous fluoride ions to react with such organophosphorus compounds. The facility of the degradation process even in dilute fluoride solutions is due to the increased reactivity of fluoride, which is caused by the significant low activation barrier for the P-F bond formation. In addition, the unique nucleophilicity of fluoride versus hydroxide toward VX, in contrast to their relative basicity, is discussed. Although the reaction outcomes were similar, much slower reaction rates were observed experimentally for the VX-model (1) in comparison to VX. PMID:23083335

  15. The Inherent Conformational Preferences of Glutamine-Containing Peptides: the Role for Side-Chain Backbone Hydrogen Bonds

    NASA Astrophysics Data System (ADS)

    Walsh, Patrick S.; McBurney, Carl; Gellman, Samuel H.; Zwier, Timothy S.

    2015-06-01

    Glutamine is widely known to be found in critical regions of peptides which readily fold into amyloid fibrils, the structures commonly associated with Alzheimer's disease and glutamine repeat diseases such as Huntington's disease. Building on previous single-conformation data on Gln-containing peptides containing an aromatic cap on the N-terminus (Z-Gln-OH and Z-Gln-NHMe), we present here single-conformation UV and IR spectra of Ac-Gln-NHBn and Ac-Ala-Gln-NHBn, with its C-terminal benzyl cap. These results point towards side-chain to backbone hydrogen bonds dominating the structures observed in the cold, isolated environment of a molecular beam. We have identified and assigned three main conformers for Ac-Gln-NHBn all involving primary side-chain to backbone interactions. Ac-Ala-Gln-NHBn extends the peptide chain by one amino acid, but affords an improvement in the conformational flexibility. Despite this increase in the flexibility, only a single conformation is observed in the gas-phase: a structure which makes use of both side-chain-to-backbone and backbone-to-backbone hydrogen bonds.

  16. A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase

    PubMed Central

    Sträter, Norbert; Sun, Lee; Kantrowitz, E. R.; Lipscomb, William N.

    1999-01-01

    The active sites of aminopeptidase A (PepA) from Escherichia coli and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 Å and 1.6 Å resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions. It is shown that PepA is activated about 10-fold by bicarbonate when l-leucine p-nitroanilide is used as a substrate. No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases. A mutational analysis shows that Arg-356 influences activity by binding the bicarbonate ion but is not essential for activity. Mutation of the catalytic Lys-282 reduces kcat/Km about 10,000-fold. PMID:10500145

  17. Mechanistic Examination of Cβ–Cγ Bond Cleavages of Tryptophan Residues during Dissociations of Molecular Peptide Radical Cations

    SciTech Connect

    Song, Tao; Ma, Ching-Yung; Chu, Ivan K.; Siu, Chi-Kit; Laskin, Julia

    2013-02-14

    In this study, we used collision-induced dissociation (CID) to examine the gas-phase fragmentations of [GnW]•+ (n = 2-4) and [GXW]•+ (X = C, S, L, F, Y, Q) species. The Cβ–Cγ bond cleavage of a C-terminal decarboxylated tryptophan residue ([M - CO2]•+) can generate [M - CO2 - 116]+, [M - CO2 - 117]•+, and [1H-indole]•+ (m/z 117) species as possible product ions. Competition between the formation of [M - CO2 - 116]+ and [1H-indole]•+ systems implies the existence of a proton-bound dimer formed between the indole ring and peptide backbone. Formation of such a proton-bound dimer is facile via a protonation of the tryptophan γ-carbon atom as suggested by density functional theory (DFT) calculations. DFT calculations also suggested the initially formed ion 2--the decarboxylated species that is active against Cβ–Cγ bond cleavage -can efficiently isomerize to form a more-stable -radical isomer (ion 9) as supported by Rice-Ramsperger-Kassel-Marcus (RRKM) modeling. The Cβ–Cγ bond cleavage of a tryptophan residue also can occur directly from peptide radical cations containing a basic residue. CID of [WGnR]•+ (n = 1-3) radical cations consistently resulted in predominant formation of [M-116]+ product ions. It appears that the basic arginine residue tightly sequesters the proton and allows the charge-remote Cβ–Cγ bond cleavage to prevail over the charge-directed one. DFT calculations predicted the barrier for the former is 6.2 kcal mol -1 lower than that of the latter. Furthermore, the pathway involving a salt-bridge intermediate also was accessible during such a bond cleavage event.

  18. Oscillator strength of the peptide bond {pi}* resonances at all relevant x-ray absorption edges

    SciTech Connect

    Kummer, K.; Vyalikh, D. V.; Molodtsov, S. L.; Sivkov, V. N.; Nekipelov, S. V.; Maslyuk, V. V.; Mertig, I.; Blueher, A.; Mertig, M.; Bredow, T.

    2009-10-15

    Absolute x-ray absorption cross sections of a regular bacterial surface-layer protein deposited on a naturally oxidized silicon substrate were determined experimentally. Upon separation of the partial cross sections of the three relevant 1s absorption edges, the oscillator strengths of the 1s{yields}{pi}* excitations within the peptide-backbone unit were extracted. Comparison with results of first-principles calculations revealed their close correlation to the topology of {pi}{sub peptide}* orbitals of the peptide backbone.

  19. Methyl group dynamics in paracetamol and acetanilide: probing the static properties of intermolecular hydrogen bonds formed by peptide groups

    NASA Astrophysics Data System (ADS)

    Johnson, M. R.; Prager, M.; Grimm, H.; Neumann, M. A.; Kearley, G. J.; Wilson, C. C.

    1999-06-01

    Measurements of tunnelling and librational excitations for the methyl group in paracetamol and tunnelling excitations for the methyl group in acetanilide are reported. In both cases, results are compared with molecular mechanics calculations, based on the measured low temperature crystal structures, which follow an established recipe. Agreement between calculated and measured methyl group observables is not as good as expected and this is attributed to the presence of comprehensive hydrogen bond networks formed by the peptide groups. Good agreement is obtained with a periodic quantum chemistry calculation which uses density functional methods, these calculations confirming the validity of the one-dimensional rotational model used and the crystal structures. A correction to the Coulomb contribution to the rotational potential in the established recipe using semi-emipircal quantum chemistry methods, which accommodates the modified charge distribution due to the hydrogen bonds, is investigated.

  20. Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling.

    PubMed

    Liu, Baoyu; Chen, Wei; Evavold, Brian D; Zhu, Cheng

    2014-04-10

    TCR-pMHC interactions initiate adaptive immune responses, but the mechanism of how such interactions under force induce T cell signaling is unclear. We show that force prolongs lifetimes of single TCR-pMHC bonds for agonists (catch bonds) but shortens those for antagonists (slip bonds). Both magnitude and duration of force are important, as the highest Ca(2+) responses were induced by 10 pN via both pMHC catch bonds whose lifetime peaks at this force and anti-TCR slip bonds whose maximum lifetime occurs at 0 pN. High Ca(2+) levels require early and rapid accumulation of bond lifetimes, whereas short-lived bonds that slow early accumulation of lifetimes correspond to low Ca(2+) responses. Our data support a model in which force on the TCR induces signaling events depending on its magnitude, duration, frequency, and timing, such that agonists form catch bonds that trigger the T cell digitally, whereas antagonists form slip bonds that fail to activate. PMID:24725404

  1. Pulling out a peptide chain from -sheet crystallite: Propagation of instability of H-bonds under shear force

    NASA Astrophysics Data System (ADS)

    Xu, Changjian; Li, Dechang; Cheng, Yuan; Liu, Ming; Zhang, Yongwei; Ji, Baohua

    2015-06-01

    Anti-parallel -sheet crystallite as the main component of silk fibroin has attracted much attention due to its superior mechanical properties. In this study, we examine the processes of pulling a peptide chain from -sheet crystallite using steered molecular dynamics simulations to investigate the rupture behavior of the crystallite. We show that the failure of -sheet crystallite was accompanied by a propagation of instability of hydrogen-bonds (H-bonds) in the crystallite. In addition, we find that there is an optimum size of the crystallite at which the H-bonds can work cooperatively to achieve the highest shear strength. In addition, we find that the stiffness of loading device and the loading rates have significant effects on the rupture behavior of -sheet crystallite. The stiff loading device facilitates the rebinding of the H-bond network in the stick-slip motion between the chains, while the soft one suppresses it. Moreover, the rupture force of -sheet crystallites decreases with loading rate. Particularly, when the loading rate decreases to a critical value, the rupture force of the -sheet crystallite becomes independent of the loading rates. This study provides atomistic details of rupture behaviors of -sheet crystallite, and, therefore, sheds valuable light on the underlying mechanism of the superior mechanical properties of silk fibroin.

  2. Cloning, sequencing and overexpression of the gene for prokaryotic factor EF-P involved in peptide bond synthesis.

    PubMed Central

    Aoki, H; Adams, S L; Chung, D G; Yaguchi, M; Chuang, S E; Ganoza, M C

    1991-01-01

    A soluble protein EF-P (elongation factor P) from Escherichia coli has been purified and shown to stimulate efficient translation and peptide-bond synthesis on native or reconstituted 70S ribosomes in vitro. Based on the partial amino acid sequence of EF-P, 18- and 24-nucleotide DNA probes were synthesized and used to screen lambda phage clones from the Kohara Gene Bank. The entire EF-P gene was detected on lambda clone #650 which contains sequences from the 94 minute region of the E.coli genome. Two DNA fragments, 3.0 and 0.78 kilobases in length encompassing the gene, were isolated and cloned into pUC18 and pUC19. Partially purified extracts from cells transformed with these plasmids overrepresented a protein which co-migrates with EF-P upon SDS polyacrylamide gel electrophoresis, and also exhibited increased EF-P mediated peptide-bond synthetic activity. Based on DNA sequence analysis of this gene, the EF-P protein consists of 187 amino acids with a calculated molecular weight of 20,447. The sequence and chromosomal location of EF-P establishes it as a unique gene product. Images PMID:1956781

  3. Formation of the First Peptide Bond: The Structure of EF-P Bound to the 70S Ribosome

    SciTech Connect

    Blaha, Gregor; Stanley, Robin E.; Steitz, Thomas A.; Yale

    2009-10-21

    Elongation factor P (EF-P) is an essential protein that stimulates the formation of the first peptide bond in protein synthesis. Here we report the crystal structure of EF-P bound to the Thermus thermophilus 70S ribosome along with the initiator transfer RNA N-formyl-methionyl-tRNAi (fMet-tRNA{sub i}{sup fMet}) and a short piece of messenger RNA (mRNA) at a resolution of 3.5 angstroms. EF-P binds to a site located between the binding site for the peptidyl tRNA (P site) and the exiting tRNA (E site). It spans both ribosomal subunits with its amino-terminal domain positioned adjacent to the aminoacyl acceptor stem and its carboxyl-terminal domain positioned next to the anticodon stem-loop of the P site-bound initiator tRNA. Domain II of EF-P interacts with the ribosomal protein L1, which results in the largest movement of the L1 stalk that has been observed in the absence of ratcheting of the ribosomal subunits. EF-P facilitates the proper positioning of the fMet-tRNA{sub i}{sup fMet} for the formation of the first peptide bond during translation initiation.

  4. Formation of the First Peptid Bond: the Structure of EF-P Bound to the 70S Ribosome

    SciTech Connect

    Blaha, G.; Stanley, R; Steitz, T

    2009-01-01

    Elongation factor P (EF-P) is an essential protein that stimulates the formation of the first peptide bond in protein synthesis. Here we report the crystal structure of EF-P bound to the Thermus thermophilus 70S ribosome along with the initiator transfer RNA N-formyl-methionyl-tRNAi (fMet-tRNAifMet) and a short piece of messenger RNA (mRNA) at a resolution of 3.5 angstroms. EF-P binds to a site located between the binding site for the peptidyl tRNA (P site) and the exiting tRNA (E site). It spans both ribosomal subunits with its amino-terminal domain positioned adjacent to the aminoacyl acceptor stem and its carboxyl-terminal domain positioned next to the anticodon stem-loop of the P site-bound initiator tRNA. Domain II of EF-P interacts with the ribosomal protein L1, which results in the largest movement of the L1 stalk that has been observed in the absence of ratcheting of the ribosomal subunits. EF-P facilitates the proper positioning of the fMet-tRNAifMet for the formation of the first peptide bond during translation initiation.

  5. Cysteine-Selective Peptide Identification: Selenium-Based Chromophore for Selective S-Se Bond Cleavage with 266 nm Ultraviolet Photodissociation.

    PubMed

    Parker, W Ryan; Holden, Dustin D; Cotham, Victoria C; Xu, Hua; Brodbelt, Jennifer S

    2016-07-19

    The tremendous number of peptides identified in current bottom-up mass spectrometric workflows, although impressive for high-throughput proteomics, results in little selectivity for more targeted applications. We describe a strategy for cysteine-selective proteomics based on a tagging method that installs a S-Se bond in peptides that is cleavable upon 266 nm ultraviolet photodissociation (UVPD). The alkylating reagent, N-(phenylseleno)phthalimide (NPSP), reacts with free thiols in cysteine residues and attaches a chromogenic benzeneselenol (SePh) group. Upon irradiation of tagged peptides with 266 nm photons, the S-Se bond is selectively cleaved, releasing a benzeneselenol moiety corresponding to a neutral loss of 156 Da per cysteine. Herein we demonstrate a new MS/MS scan mode, UVPDnLossCID, which facilitates selective screening of cysteine-containing peptides. A "prescreening" event occurs by activation of the top N peptide ions by 266 nm UVPD. Peptides exhibiting a neutral loss corresponding to one or more SePh groups are reactivated and sequenced by CID. Because of the low frequency of cysteine in the proteome, unique cysteine-containing peptides may serve as surrogates for entire proteins. UVPDnLossCID does not generate as many peptide spectrum matches (PSMs) as conventional bottom-up methods; however, UVPDnLossCID provides far greater selectivity. PMID:27320857

  6. Phosphate Triester Hydrolysis Promoted by an N2S (thiolate) Zinc Complex: Mechanistic Implications for the Metal-Dependent Reactivity of Peptide Deformylase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The zinc(II) complex (PATH)ZnOH, where PATH is an N2S(thiolate) ligand, has been investigated for its ability to promote the hydrolysis of the phosphate triester tris(4-nitrophenyl) phosphate (TNP). The hydrolysis of TNP was examined as a function of PATH-zinc(II) complex concentration, substrate co...

  7. Large Scale Solid Phase Synthesis of Peptide Drugs: Use of Commercial Anion Exchange Resin as Quenching Agent for Removal of Iodine during Disulphide Bond Formation

    PubMed Central

    Reddy, K. M. Bhaskara; Kumari, Y. Bharathi; Mallikharjunasarma, Dokka; Bulliraju, Kamana; Sreelatha, Vanjivaka; Ananda, Kuppanna

    2012-01-01

    The S-acetamidomethyl (Acm) or trityl (Trt) protecting groups are widely used in the chemical synthesis of peptides that contain one or more disulfide bonds. Treatment of peptides containing S-Acm protecting group with iodine results in simultaneous removal of the sulfhydryl protecting group and disulfide formation. However, the excess iodine needs to be quenched or adsorbed as quickly as possible after completion of the disulfide bond formation in order to minimize side reactions that are often associated with the iodination step. We report here a simple method for simultaneous quenching and removal of iodine and isolation of disulphide bridge peptides. The use of excess inexpensive anion exchange resin to the oxidized peptide from the aqueous acetic acid/methanol solution affords quantitative removal of iodine and other color impurities. This improves the resin life time of expensive chromatography media that is used in preparative HPLC column during the purification of peptide using preparative HPLC. Further, it is very useful for the conversion of TFA salt to acetate in situ. It was successfully applied commercially, to the large scale synthesis of various peptides including Desmopressin, Oxytocin, and Octreotide. This new approach offers significant advantages such as more simple utility, minimal side reactions, large scale synthesis of peptide drugs, and greater cost effectiveness. PMID:23118772

  8. Peptide bond-forming reagents HOAt and HATU are not mutagenic in the bacterial reverse mutation test.

    PubMed

    Nicolette, John; Neft, Robin E; Vanosdol, Jessica; Murray, Joel

    2016-04-01

    The peptide bond-forming reagents 1-hydroxy-7-azabenzotriazole (HOAt, CAS 39968-33-7) and O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, CAS 148893-10-1) either have structural alerts, unclassified features or are considered out of domain when evaluated for potential mutagenicity with in silico programs DEREK and CaseUltra. Since they are commonly used reagents in pharmaceutical drug syntheses, they may become drug substance or drug product impurities and would need to be either controlled to appropriately safe levels or tested for mutagenicity. Both reagents were tested in the bacterial reverse mutation (Ames) test at Covance, under GLP conditions, following the OECD test guideline and ICH S2(R1) recommendations and found to be negative. Our data show that HOAt and HATU-common pharmaceutical synthesis reagents-are not mutagenic, and can be treated as ordinary drug impurities. PMID:26840011

  9. Orthogonal halogen and hydrogen bonds involving a peptide bond model† †Electronic supplementary information (ESI) available: Experimental part, DSC, IR spectroscopic and crystallographic data. CCDC 899779–899785. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ce01514b Click here for additional data file. Click here for additional data file.

    PubMed Central

    Vasylyeva, Vera; Nayak, Susanta K.; Cavallo, Gabriella; Resnati, Giuseppe

    2014-01-01

    The peptide bond model N-methylacetamide self-assembles with a range of dihalotetrafluorobenzenes forming co-crystals that all show the occurrence of orthogonal hydrogen and halogen bonds. PMID:25663816

  10. Conformations of Prolyl-Peptide Bonds in the Bradykinin 1-5 Fragment in Solution and in the Gas Phase.

    PubMed

    Voronina, Liudmila; Masson, Antoine; Kamrath, Michael; Schubert, Franziska; Clemmer, David; Baldauf, Carsten; Rizzo, Thomas

    2016-07-27

    The dynamic nature of intrinsically disordered peptides makes them a challenge to characterize by solution-phase techniques. In order to gain insight into the relation between the disordered state and the environment, we explore the conformational space of the N-terminal 1-5 fragment of bradykinin (BK[1-5](2+)) in the gas phase by combining drift tube ion mobility, cold-ion spectroscopy, and first-principles simulations. The ion-mobility distribution of BK[1-5](2+) consists of two well-separated peaks. We demonstrate that the conformations within the peak with larger cross-section are kinetically trapped, while the more compact peak contains low-energy structures. This is a result of cis-trans isomerization of the two prolyl-peptide bonds in BK[1-5](2+). Density-functional theory calculations reveal that the compact structures have two very different geometries with cis-trans and trans-cis backbone conformations. Using the experimental CCSs to guide the conformational search, we find that the kinetically trapped species have a trans-trans configuration. This is consistent with NMR measurements performed in a solution, which show that 82% of the molecules adopt a trans-trans configuration and behave as a random coil. PMID:27366919

  11. Streptothricin biosynthesis is catalyzed by enzymes related to nonribosomal peptide bond formation.

    PubMed Central

    Fernández-Moreno, M A; Vallín, C; Malpartida, F

    1997-01-01

    In a search for strains producing biocides with a wide spectrum of activity, a new strain was isolated. This strain was taxonomically characterized as Streptomyces rochei F20, and the chemical structure of the bioactive product extracted from its fermentation broth was determined to be a mixture of streptothricins. From a genomic library of the producer strain prepared in the heterologous host Streptomyces lividans, a 7.2-kb DNA fragment which conferred resistance to the antibiotic was isolated. DNA sequencing of 5.2 kb from the cloned fragment revealed five open reading frames (ORFs) such that ORF1, -2, -3, and -4 were transcribed in the same direction while ORF5 was convergently arranged. The deduced product of ORF1 strongly resembled those of genes involved in peptide formation by a nonribosomal mechanism; the ORF2 product strongly resembled that of mphA and mphB isolated from Escherichia coli, which determines resistance to several macrolides by a macrolide 2'-phosphotransferase activity; the ORF3 product had similarities with several hydrolases; and the ORF5 product strongly resembled streptothricin acetyltransferases from different gram-positive and gram-negative bacteria. ORF5 was shown to be responsible for acetyl coenzyme A-dependent streptothricin acetylation. No similarities in the databases for the ORF4 product were found. Unlike other peptide synthases, that for streptothricin biosynthesis was arranged as a multienzymatic system rather than a multifunctional protein. Insertional inactivation of ORF1 and ORF2 (and to a lesser degree, of ORF3) abolishes antibiotic biosynthesis, suggesting their involvement in the streptothricin biosynthetic pathway. PMID:9371436

  12. Lisdexamfetamine prodrug activation by peptidase-mediated hydrolysis in the cytosol of red blood cells.

    PubMed

    Sharman, Johannah; Pennick, Michael

    2014-01-01

    Lisdexamfetamine dimesylate (LDX) is approved as a once-daily treatment for attention-deficit/hyperactivity disorder in children, adolescents, and adults in some countries. LDX is a prodrug comprising d-amphetamine covalently linked to l-lysine via a peptide bond. Following oral administration, LDX is rapidly taken up from the small intestine by active carrier-mediated transport, probably via peptide transporter 1. Enzymatic hydrolysis of the peptide bond to release d-amphetamine has previously been shown to occur in human red blood cells but not in several other tissues. Here, we report that LDX hydrolytic activity resides in human red blood cell lysate and cytosolic extract but not in the membrane fraction. Among several inhibitors tested, a protease inhibitor cocktail, bestatin, and ethylenediaminetetra-acetic acid each potently inhibited d-amphetamine production from LDX in cytosolic extract. These results suggest that an aminopeptidase is responsible for hydrolytic cleavage of the LDX peptide bond, although purified recombinant aminopeptidase B was not able to release d-amphetamine from LDX in vitro. The demonstration that aminopeptidase-like activity in red blood cell cytosol is responsible for the hydrolysis of LDX extends our understanding of the smooth and consistent systemic delivery of d-amphetamine by LDX and the long daily duration of efficacy of the drug in relieving the symptoms of attention-deficit/hyperactivity disorder. PMID:25489246

  13. Lisdexamfetamine prodrug activation by peptidase-mediated hydrolysis in the cytosol of red blood cells

    PubMed Central

    Sharman, Johannah; Pennick, Michael

    2014-01-01

    Lisdexamfetamine dimesylate (LDX) is approved as a once-daily treatment for attention-deficit/hyperactivity disorder in children, adolescents, and adults in some countries. LDX is a prodrug comprising d-amphetamine covalently linked to l-lysine via a peptide bond. Following oral administration, LDX is rapidly taken up from the small intestine by active carrier-mediated transport, probably via peptide transporter 1. Enzymatic hydrolysis of the peptide bond to release d-amphetamine has previously been shown to occur in human red blood cells but not in several other tissues. Here, we report that LDX hydrolytic activity resides in human red blood cell lysate and cytosolic extract but not in the membrane fraction. Among several inhibitors tested, a protease inhibitor cocktail, bestatin, and ethylenediaminetetra-acetic acid each potently inhibited d-amphetamine production from LDX in cytosolic extract. These results suggest that an aminopeptidase is responsible for hydrolytic cleavage of the LDX peptide bond, although purified recombinant aminopeptidase B was not able to release d-amphetamine from LDX in vitro. The demonstration that aminopeptidase-like activity in red blood cell cytosol is responsible for the hydrolysis of LDX extends our understanding of the smooth and consistent systemic delivery of d-amphetamine by LDX and the long daily duration of efficacy of the drug in relieving the symptoms of attention-deficit/hyperactivity disorder. PMID:25489246

  14. {{text{C}}_{α }} - {text{C}} Bond Cleavage of the Peptide Backbone in MALDI In-Source Decay Using Salicylic Acid Derivative Matrices

    NASA Astrophysics Data System (ADS)

    Asakawa, Daiki; Takayama, Mitsuo

    2011-07-01

    The use of 5-formylsalicylic acid (5-FSA) and 5-nitrosalicylic acid (5-NSA) as novel matrices for in-source decay (ISD) of peptides in matrix-assisted laser desorption/ionization (MALDI) is described. The use of 5-FSA and 5-NSA generated a- and x-series ions accompanied by oxidized peptides [M - 2 H + H]+. The preferential formation of a- and x-series ions was found to be dependent on the hydrogen-accepting ability of matrix. The hydrogen-accepting ability estimated from the ratio of signal intensity of oxidized product [M - 2 H + H]+ to that of non-oxidized protonated molecule [M + H]+ of peptide was of the order 5-NSA > 5-FSA > 5-aminosalicylic acid (5-ASA) ≒ 2,5-dihydroxyl benzoic acid (2,5-DHB) ≒ 0. The results suggest that the hydrogen transfer reaction from peptide to 5-FSA and 5-NSA occurs during the MALDI-ISD processes. The hydrogen abstraction from peptides results in the formation of oxidized peptides containing a radical site on the amide nitrogen with subsequent radical-induced cleavage at the {{{C}}_{α }} - {{C}} bond, leading to the formation of a- and x-series ions. The most significant feature of MALDI-ISD with 5-FSA and 5-NSA is the specific cleavage of the {{{C}}_{α }} - {{C}} bond of the peptide backbone without degradation of side-chain and post-translational modifications (PTM). The matrix provides a useful complementary method to conventional MALDI-ISD for amino acid sequencing and site localization of PTMs in peptides.

  15. Enhancement of nano-hydroxyapatite bonding to dentin through a collagen/calcium dual-affinitive peptide for dentinal tubule occlusion.

    PubMed

    Wang, Ronghan; Wang, Qiong; Wang, Xiumei; Tian, Lili; Liu, Huiying; Zhao, Miaomiao; Peng, Ce; Cai, Qiang; Shi, Yunming

    2014-02-01

    Calcium phosphate crystals, as the main component of dentin and enamel, have been widely used for the occlusion of dentinal tubules. However, the low bond strength and poor sealing effect limit their clinical practicality. In this study, a collagen/calcium dual-affinitive peptide E8DS (EEEEEEEEDSpESpSpEEDR) and nano-hydroxyapatite (nano-HAp) flocculi were developed to seal dentin tubules for reducing dentin hypersensitivity, whereas the E8DS peptides were pre-applied to improve the adhesion of occlusive hydroxyapatite coating on dentin collagen matrix for the long-lasting sealing effect and relief from hypersensitivity. Our study showed that E8DS peptides had a strong affinity with dentin collageneous matrix that almost 43.7% of initial E8DS peptides immobilized on exposed dentin samples remained detained after continuous washing by distilled water for four weeks at a rate of 1 mL/min. Nano-HAp flocculi were obtained by re-neutralization of HAp-HCl solution and then brushed onto the surfaces of pre-treated human dentin disks with E8DS peptides, which showed a perfect occlusion of exposed dentinal tubules, as compared with Nano-HAp only and a commercial desensitizer, Green Or. With only around 10-min E8DS peptide pre-treatment, the occlusive mineral layers remained intact against consecutively stirred washing in phosphate-buffered saline or coke for 15 min, and 6 min of tooth-brushing, which implied that our E8DS peptide could comparatively improve the durability of sealant-dentin interface bonds for long-lasting dentine desensitization. PMID:24505078

  16. Plane wave density functional theory studies of the structural and the electronic properties of amino acids attached to graphene oxide via peptide bonding

    NASA Astrophysics Data System (ADS)

    Min, Byeong June; Jeong, Hae Kyung; Lee, ChangWoo

    2015-08-01

    We studied via plane wave pseudopotential total-energy calculations within the local spin density approximation (LSDA) the electronic and the structural properties of amino acids (alanine, glycine, and histidine) attached to graphene oxide (GO) by peptide bonding. The HOMO-LUMO gap, the Hirshfeld charges, and the equilibrium geometrical structures exhibit distinctive variations that depend on the species of the attached amino acid. The GO-amino acid system appears to be a good candidate for a biosensor.

  17. Combined use of ion mobility and collision-induced dissociation to investigate the opening of disulfide bridges by electron-transfer dissociation in peptides bearing two disulfide bonds.

    PubMed

    Massonnet, Philippe; Upert, Gregory; Smargiasso, Nicolas; Gilles, Nicolas; Quinton, Loïc; De Pauw, Edwin

    2015-01-01

    Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability toward enzymatic degradations and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e., the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electron-transfer dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides, resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion mobility, and (iii) to characterize the species using collision-induced dissociation (CID). Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH· radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process. PMID:25915795

  18. Peptide bonds affect the formation of haloacetamides, an emerging class of N-DBPs in drinking water: free amino acids versus oligopeptides.

    PubMed

    Chu, Wenhai; Li, Xin; Gao, Naiyun; Deng, Yang; Yin, Daqiang; Li, Dongmei; Chu, Tengfei

    2015-01-01

    Haloacetamides (HAcAms), an emerging class of nitrogenous disinfection by-products (N-DBPs) of health concern, have been frequently identified in drinking waters. It has long been appreciated that free amino acids (AAs), accounting for a small fraction of the dissolved organic nitrogen (DON) pool, can form dichloroacetamide (DCAcAm) during chlorination. However, the information regarding the impacts of combined AAs, which contribute to the greatest identifiable DON portion in natural waters, is limited. In this study, we compared the formation of HAcAms from free AAs (tyrosine [Tyr] and alanine [Ala]) and combined AAs (Tyr-Ala, Ala-Tyr, Tyr-Tyr-Tyr, Ala-Ala-Ala), and found that HAcAm formation from the chlorination of AAs in combined forms (oligopeptides) significantly exhibited a different pattern with HAcAm formation from free AAs. Due to the presence of peptide bonds in tripeptides, Tyr-Tyr-Tyr and Ala-Ala-Ala produced trichloroacetamide (TCAcAm) in which free AAs was unable to form TCAcAm during chlorination. Moreover, peptide bond in tripeptides formed more tri-HAcAms than di-HAcAms in the presence of bromide. Therefore, the peptide bond may be an important indicator to predict the formation of specific N-DBPs in chlorination. The increased use of algal- and wastewater-impacted water as drinking water sources will increase health concerns over exposure to HAcAms in drinking water. PMID:26394759

  19. Peptide bonds affect the formation of haloacetamides, an emerging class of N-DBPs in drinking water: free amino acids versus oligopeptides

    PubMed Central

    Chu, Wenhai; Li, Xin; Gao, Naiyun; Deng, Yang; Yin, Daqiang; Li, Dongmei; Chu, Tengfei

    2015-01-01

    Haloacetamides (HAcAms), an emerging class of nitrogenous disinfection by-products (N-DBPs) of health concern, have been frequently identified in drinking waters. It has long been appreciated that free amino acids (AAs), accounting for a small fraction of the dissolved organic nitrogen (DON) pool, can form dichloroacetamide (DCAcAm) during chlorination. However, the information regarding the impacts of combined AAs, which contribute to the greatest identifiable DON portion in natural waters, is limited. In this study, we compared the formation of HAcAms from free AAs (tyrosine [Tyr] and alanine [Ala]) and combined AAs (Tyr-Ala, Ala-Tyr, Tyr-Tyr-Tyr, Ala-Ala-Ala), and found that HAcAm formation from the chlorination of AAs in combined forms (oligopeptides) significantly exhibited a different pattern with HAcAm formation from free AAs. Due to the presence of peptide bonds in tripeptides, Tyr-Tyr-Tyr and Ala-Ala-Ala produced trichloroacetamide (TCAcAm) in which free AAs was unable to form TCAcAm during chlorination. Moreover, peptide bond in tripeptides formed more tri-HAcAms than di-HAcAms in the presence of bromide. Therefore, the peptide bond may be an important indicator to predict the formation of specific N-DBPs in chlorination. The increased use of algal- and wastewater-impacted water as drinking water sources will increase health concerns over exposure to HAcAms in drinking water. PMID:26394759

  20. Hydrolysis of Sequenced β-Casein Peptides Provides New Insight into Peptidase Activity from Thermophilic Lactic Acid Bacteria and Highlights Intrinsic Resistance of Phosphopeptides

    PubMed Central

    Deutsch, Stéphanie-Marie; Molle, Daniel; Gagnaire, Valérie; Piot, Michel; Atlan, Danièle; Lortal, Sylvie

    2000-01-01

    The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified β-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the β-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the β-casein hydrolysate as the substrate at pH 5.5 and 24°C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing β-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the β-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species. PMID:11097915

  1. Disruption of Hydrogen Bonds between Major Histocompatibility Complex Class II and the Peptide N-Terminus Is Not Sufficient to Form a Human Leukocyte Antigen-DM Receptive State of Major Histocompatibility Complex Class II

    PubMed Central

    Schulze, Monika-Sarah E. D.; Anders, Anne-Kathrin; Sethi, Dhruv K.; Call, Melissa J.

    2013-01-01

    Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306–318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue. PMID:23976922

  2. Guanylyl cyclase/natriuretic peptide receptor-A signaling antagonizes phosphoinositide hydrolysis, Ca2+ release, and activation of protein kinase C

    PubMed Central

    Pandey, Kailash N.

    2014-01-01

    Thus far, three related natriuretic peptides (NPs) and three distinct sub-types of cognate NP receptors have been identified and characterized based on the specific ligand binding affinities, guanylyl cyclase activity, and generation of intracellular cGMP. Atrial and brain natriuretic peptides (ANP and BNP) specifically bind and activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), and C-type natriuretic peptide (CNP) shows specificity to activate guanylyl cyclase/natriuretic peptide receptor-B (GC-B/NPRB). All three NPs bind to natriuretic peptide receptor-C (NPRC), which is also known as clearance or silent receptor. The NPRA is considered the principal biologically active receptor of NP family; however, the molecular signaling mechanisms of NP receptors are not well understood. The activation of NPRA and NPRB produces the intracellular second messenger cGMP, which serves as the major signaling molecule of all three NPs. The activation of NPRB in response to CNP also produces the intracellular cGMP; however, at lower magnitude than that of NPRA, which is activated by ANP and BNP. In addition to enhanced accumulation of intracellular cGMP in response to all three NPs, the levels of cAMP, Ca2+ and inositol triphosphate (IP3) have also been reported to be altered in different cells and tissue types. Interestingly, ANP has been found to lower the concentrations of cAMP, Ca2+, and IP3; however, NPRC has been proposed to increase the levels of these metabolic signaling molecules. The mechanistic studies of decreased and/or increased levels of cAMP, Ca2+, and IP3 in response to NPs and their receptors have not yet been clearly established. This review focuses on the signaling mechanisms of ANP/NPRA and their biological effects involving an increased level of intracellular accumulation of cGMP and a decreased level of cAMP, Ca2+, and IP3 in different cells and tissue systems. PMID:25202235

  3. Unusual folding propensity of an unsubstituted β,γ-hybrid model peptide: importance of the C-H⋅⋅⋅O intramolecular hydrogen bond.

    PubMed

    Venugopalan, Paloth; Kishore, Raghuvansh

    2013-07-22

    The single-crystal X-ray diffraction analysis of a β,γ-hybrid model peptide Boc-β-Ala-γ-Abu-NH2 revealed the existence of four crystallographically independent molecules (A, B, C and D conformers) in the asymmetric unit. The analysis revealed that unusual β-turn-like folded structures predominate, wherein the conformational space of non-proteinogenic β-Ala and γ-Abu residues are restricted to gauche-gauche-skew and skew-gauche-trans-skew orientations, respectively. Interestingly, the U-shaped conformers are seemingly stabilised by an effective unconventional C-H⋅⋅⋅O intramolecular hydrogen bond, encompassing a non-covalent 14-membered ring-motif. Taking into account the signs of torsion angles, these conformers could be grouped into two distinct categories, A/B and C/D, establishing the incidence of non-superimposable stereogeometrical features across a non-chiral one-component peptide model system, that is, "mirror-image-like" relationships. The natural occurrence of β-Ala and γ-Abu entities in various pharmacologically important molecules, coupled with their biocompatibilities, highlight how the non-functionalised β,γ-hybrid segment may offer unique advantages for introducing and/or manipulating a wide spectrum of biologically relevant hydrogen bonded secondary structural mimics in short synthetic peptides. PMID:23775881

  4. Un-catalyzed peptide bond formation between two monomers of glycine, alanine, serine, threonine, and aspartic acid in gas phase: a density functional theory study

    NASA Astrophysics Data System (ADS)

    Bhunia, Snehasis; Singh, Ajeet; Ojha, Animesh K.

    2016-05-01

    In the present report, un-catalyzed peptide bond formation between two monomers of glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), and aspartic acid (Asp) has been investigated in gas phase via two steps reaction mechanism and concerted mechanism at B3LYP/6-31G(d,p) and M062X/6-31G(d,p) level of theories. The peptide bond is formed through a nucleophilic reaction via transition states, TS1 and TS2 in stepwise mechanism. The TS1 reveals formation of a new C-N bond while TS2 illustrate the formation of C=O bond. In case of concerted mechanism, C-N bond is formed by a single four-centre transition state (TS3). The energy barrier is used to explain the involvement of energy at each step of the reaction. The energy barrier (20-48 kcal/mol) is required for the transformation of reactant state R1 to TS1 state and intermediate state I1 to TS2 state. The large value of energy barrier is explained in terms of distortion and interaction energies for stepwise mechanism. The energy barrier of TS3 in concerted mechanism is very close to the energy barrier of the first transition state (TS1) of the stepwise mechanism for the formation of Gly-Gly and Ala-Ala di- peptide. However, in case of Ser-Ser, Thr-Thr and Asp-Asp di-peptide, the energy barrier of TS3 is relatively high than that of the energy barrier of TS1 calculated at B3LYP/6-31G(d,p) and M062X/6-31G(d,p) level of theories. In both the mechanisms, the value of energy barrier calculated at B3LYP/6-31G(d,p) level of theory is greater than that of the value calculated at M062X/6-31G(d,p) level of theory.

  5. A single molecule study of cellulase hydrolysis of crystalline cellulose

    NASA Astrophysics Data System (ADS)

    Liu, Yu-San; Luo, Yonghua; Baker, John O.; Zeng, Yining; Himmel, Michael E.; Smith, Steve; Ding, Shi-You

    2010-02-01

    Cellobiohydrolase-I (CBH I), a processive exoglucanase secreted by Trichoderma reesei, is one of the key enzyme components in a commercial cellulase mixture currently used for processing biomass to biofuels. CBH I contains a family 7 glycoside hydrolase catalytic module, a family 1 carbohydrate-binding module (CBM), and a highlyglycosylated linker peptide. It has been proposed that the CBH I cellulase initiates the hydrolysis from the reducing end of one cellulose chain and successively cleaves alternate β-1,4-glycosidic bonds to release cellobiose as its principal end product. The role each module of CBH I plays in the processive hydrolysis of crystalline cellulose has yet to be convincingly elucidated. In this report, we use a single-molecule approach that combines optical (Total Internal Reflection Fluorescence microscopy, or TIRF-M) and non-optical (Atomic Force Microscopy, or AFM) imaging techniques to analyze the molecular motion of CBM tagged with green fluorescence protein (GFP), and to investigate the surface structure of crystalline cellulose and changes made in the structure by CBM and CBH I. The preliminary results have revealed a confined nanometer-scale movement of the TrCBM1-GFP bound to cellulose, and decreases in cellulose crystal size as well as increases in surface roughness during CBH I hydrolysis of crystalline cellulose.

  6. A conserved secondary structural motif in 23S rRNA defines the site of interaction of amicetin, a universal inhibitor of peptide bond formation.

    PubMed Central

    Leviev, I G; Rodriguez-Fonseca, C; Phan, H; Garrett, R A; Heilek, G; Noller, H F; Mankin, A S

    1994-01-01

    The binding site and probable site of action have been determined for the universal antibiotic amicetin which inhibits peptide bond formation. Evidence from in vivo mutants, site-directed mutations and chemical footprinting all implicate a highly conserved motif in the secondary structure of the 23S-like rRNA close to the central circle of domain V. We infer that this motif lies at, or close to, the catalytic site in the peptidyl transfer centre. The binding site of amicetin is the first of a group of functionally related hexose-cytosine inhibitors to be localized on the ribosome. Images PMID:8157007

  7. Hydrolysis of oligoribonucleotides: influence of sequence and length.

    PubMed Central

    Kierzek, R

    1992-01-01

    The chemical stability of phosphodiester bonds of some oligoribonucleotides in the presence of a cofactor like polyvinylpyrolidine (PVP) is sequence dependent. It was found that pyrimidine-A (YA) and pyrimidine-C (YC) are especially susceptible to hydrolysis. The hydrolyzability of this same phosphodiester bond is dependent on its position in the oligomer. The presence of 3' and 5'-adjacent nucleotides enhances hydrolysis of the UA phosphodiester bond. The acceleration of the hydrolysis of UA by a 5'-adjacent nucleotide is not base dependent. However, a 3'-adjacent purine increases hydrolysis of a UA phosphodiester bond more than a 3'-pyrimidine. The presence of the exoamino group on the 3'-side base (on 6 and 4 position for adenosine and cytidine, respectively) of YA or YZ phosphodiester bond is required for hydrolysis. Images PMID:1408823

  8. Identification and cleavage of breakable single bonds by selective oxidation, reduction, and hydrolysis. Annual report, October 1, 1980-September 30, 1981

    SciTech Connect

    Hirschon, A.S.; Zevely, J.; Mayo, F.R.

    1981-11-12

    The objective of this project is to determine the structure of bituminous coal by determining the proportions of the various kinds of connecting bonds and how they can best be broken. Results obtained during the past quarter are presented for the following tasks: (1) extractions and fractionations of coal products which covers pyridine extraction, fractionation of TIPS fractions, EDA extraction of Illinois No. 6 coal and swelling ratios of coal samples; (2) experiments on breakable single bonds which cover reactions of ethylenediamine and model ethers, reaction of pyridine-extracted coal with Me/sub 3/SiI, Baeyer-Villiger oxidations, reaction to diphenylmethane with 15% HNO/sub 3/, cleavage of TIPS with ZnI/sub 2/, and cleavage of black acids; and (3) oxygen oxidation No. 18. Some of the highlights of these studies are: (1) some model ethers are not cleaved by EDA under extraction conditions; (2) oxidation of diaryl ketones with m-chloroperbenzoic acid and saponification of the resulting esters in promising for identifying ketones, (3) treatment of a black acid with pyridine hydroiodide reduced the acid's molecular weight and increased its solubility in pyridine, but treatment with ZnI/sub 2/ was ineffective; (4) in comparison with 0.1 M K/sub 2/S/sub 2/O/sub 8/, 0.01 M persulfate is relatively ineffective in accelerating oxidation of BnNH/sub 2/-extracted coal in water suspension. 2 figures, 3 tables.

  9. Antimicrobial Cyclic Peptides for Plant Disease Control

    PubMed Central

    Lee, Dong Wan; Kim, Beom Seok

    2015-01-01

    Antimicrobial cyclic peptides derived from microbes bind stably with target sites, have a tolerance to hydrolysis by proteases, and a favorable degradability under field conditions, which make them an attractive proposition for use as agricultural fungicides. Antimicrobial cyclic peptides are classified according to the types of bonds within the ring structure; homodetic, heterodetic, and complex cyclic peptides, which in turn reflect diverse physicochemical features. Most antimicrobial cyclic peptides affect the integrity of the cell envelope. This is achieved through direct interaction with the cell membrane or disturbance of the cell wall and membrane component biosynthesis such as chitin, glucan, and sphingolipid. These are specific and selective targets providing reliable activity and safety for non-target organisms. Synthetic cyclic peptides produced through combinatorial chemistry offer an alternative approach to develop antimicrobials for agricultural uses. Those synthesized so far have been studied for antibacterial activity, however, the recent advancements in powerful technologies now promise to provide novel antimicrobial cyclic peptides that are yet to be discovered from natural resources. PMID:25774105

  10. Quantum chemical studies of a model for peptide bond formation. 3. Role of magnesium cation in formation of amide and water from ammonia and glycine

    NASA Technical Reports Server (NTRS)

    Oie, T.; Loew, G. H.; Burt, S. K.; MacElroy, R. D.

    1984-01-01

    The SN2 reaction between glycine and ammonia molecules with magnesium cation Mg2+ as a catalyst has been studied as a model reaction for Mg(2+)-catalyzed peptide bond formation using the ab initio Hartree-Fock molecular orbital method. As in previous studies of the uncatalyzed and amine-catalyzed reactions between glycine and ammonia, two reaction mechanisms have been examined, i.e., a two-step and a concerted reaction. The stationary points of each reaction including intermediate and transition states have been identified and free energies calculated for all geometry-optimized reaction species to determine the thermodynamics and kinetics of each reaction. Substantial decreases in free energies of activation were found for both reaction mechanisms in the Mg(2+)-catalyzed amide bond formation compared with those in the uncatalyzed and amine-catalyzed amide bond formation. The catalytic effect of the Mg2+ cation is to stabilize both the transition states and intermediate, and it is attributed to the neutralization of the developing negative charge on the electrophile and formation of a conformationally flexible nonplanar five-membered chelate ring structure.

  11. Fluoride-18 Radiolabeling of Peptides Bearing an Aminooxy Functional Group to a Prosthetic Ligand via an Oxime Bond

    PubMed Central

    Carberry, Patrick; Carpenter, Alan P.; Kung, Hank F.

    2011-01-01

    We have developed a novel F-18 prosthetic ligand named fluoro-PEG-benzaldehyde (FPBA) 1. [18F]-FPBA 1 is formed in situ from its radiolabeled precursor [18F]6. Compound 6 is efficiently synthesized in four steps starting from commercially available 6-bromo-3-pyridine carbaldehyde 2. [18F]-FPBA was evaluated as a prosthetic ligand to radiolabel three cyclic peptides bearing an aminooxy functional group at the N-terminus position. Acetal [18F]6 is purified by either solid-phase extraction (SPE) or reverse-phase HPLC with the overall radiochemical yields (RCY) and radiochemical purity (RCP) in very close agreement. The SPE purification process has the advantage of shorter reaction times (71–87 min for entire reaction sequence), while the use of the reverse-phase HPLC purification process allows the use of up to fifty times less of the expensive synthetic peptides (~ 50 nmol) in the oxime coupling reaction. We have demonstrated an efficient methodology in the production of [18F]-FPBA 1 and demonstrated its use as a prosthetic ligand for the labeling of peptides possessing an aminooxy functional group. PMID:22024031

  12. Stereoelectronic control in peptide bond formation. Ab initio calculations and speculations on the mechanism of action of serine proteases.

    PubMed Central

    Gorenstein, D G; Taira, K

    1984-01-01

    Ab initio molecular orbital calculations have been performed on the reaction profile for the addition/elimination reaction between ammonia and formic acid, proceeding via a tetrahedral intermediate: NH3 + HCO2H----H2NCH(OH)2----NH2CHO + H2O. Calculated transition state energies for the first addition step of the reaction revealed that a lone pair on the oxygen of the OH group, which is antiperiplanar to the attacking nitrogen, stabilized the transition state by 3.9 kcal/mol, thus supporting the hypothesis of stereoelectronic control for this reaction. In addition, a secondary, counterbalancing stereoelectronic effect stabilizes the second step, water elimination, transition state by 3.1 kcal/mol if the lone pair on the leaving water oxygen is not antiperiplanar to the C-N bond. The best conformation for the transition states was thus one with a lone pair antiperiplanar to the adjacent scissile bond and also one without a lone-pair orbital on the scissile bond oxygen or nitrogen antiperiplanar to the adjacent polar bond. The significance of these stereoelectronic effects for the mechanism of action of serine proteases is discussed. PMID:6394065

  13. On peptide bond formation, translocation, nascent protein progression and the regulatory properties of ribosomes. Derived on 20 October 2002 at the 28th FEBS Meeting in Istanbul.

    PubMed

    Agmon, Ilana; Auerbach, Tamar; Baram, David; Bartels, Heike; Bashan, Anat; Berisio, Rita; Fucini, Paola; Hansen, Harly A S; Harms, Joerg; Kessler, Maggie; Peretz, Moshe; Schluenzen, Frank; Yonath, Ada; Zarivach, Raz

    2003-06-01

    High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a two-fold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3' end around a local two-fold rotation axis, identified within the PTC. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation. PMID:12787020

  14. An efficient PEGylated liposomal nanocarrier containing cell-penetrating peptide and pH-sensitive hydrazone bond for enhancing tumor-targeted drug delivery.

    PubMed

    Ding, Yuan; Sun, Dan; Wang, Gui-Ling; Yang, Hong-Ge; Xu, Hai-Feng; Chen, Jian-Hua; Xie, Ying; Wang, Zhi-Qiang

    2015-01-01

    Cell-penetrating peptides (CPPs) as small molecular transporters with abilities of cell penetrating, internalization, and endosomal escape have potential prospect in drug delivery systems. However, a bottleneck hampering their application is the poor specificity for cells. By utilizing the function of hydration shell of polyethylene glycol (PEG) and acid sensitivity of hydrazone bond, we constructed a kind of CPP-modified pH-sensitive PEGylated liposomes (CPPL) to improve the selectivity of these peptides for tumor targeting. In CPPL, CPP was directly attached to liposome surfaces via coupling with stearate (STR) to avoid the hindrance of PEG as a linker on the penetrating efficiency of CPP. A PEG derivative by conjugating PEG with STR via acid-degradable hydrazone bond (PEG2000-Hz-STR, PHS) was synthesized. High-performance liquid chromatography and flow cytometry demonstrated that PHS was stable at normal neutral conditions and PEG could be completely cleaved from liposome surface to expose CPP under acidic environments in tumor. An optimal CPP density on liposomes was screened to guaranty a maximum targeting efficiency on tumor cells as well as not being captured by normal cells that consequently lead to a long circulation in blood. In vitro and in vivo studies indicated, in 4 mol% CPP of lipid modified system, that CPP exerted higher efficiency on internalizing the liposomes into targeted subcellular compartments while remaining inactive and free from opsonins at a maximum extent in systemic circulation. The 4% CPPL as a drug delivery system will have great potential in the clinical application of anticancer drugs in future. PMID:26491292

  15. An efficient PEGylated liposomal nanocarrier containing cell-penetrating peptide and pH-sensitive hydrazone bond for enhancing tumor-targeted drug delivery

    PubMed Central

    Ding, Yuan; Sun, Dan; Wang, Gui-Ling; Yang, Hong-Ge; Xu, Hai-Feng; Chen, Jian-Hua; Xie, Ying; Wang, Zhi-Qiang

    2015-01-01

    Cell-penetrating peptides (CPPs) as small molecular transporters with abilities of cell penetrating, internalization, and endosomal escape have potential prospect in drug delivery systems. However, a bottleneck hampering their application is the poor specificity for cells. By utilizing the function of hydration shell of polyethylene glycol (PEG) and acid sensitivity of hydrazone bond, we constructed a kind of CPP-modified pH-sensitive PEGylated liposomes (CPPL) to improve the selectivity of these peptides for tumor targeting. In CPPL, CPP was directly attached to liposome surfaces via coupling with stearate (STR) to avoid the hindrance of PEG as a linker on the penetrating efficiency of CPP. A PEG derivative by conjugating PEG with STR via acid-degradable hydrazone bond (PEG2000-Hz-STR, PHS) was synthesized. High-performance liquid chromatography and flow cytometry demonstrated that PHS was stable at normal neutral conditions and PEG could be completely cleaved from liposome surface to expose CPP under acidic environments in tumor. An optimal CPP density on liposomes was screened to guaranty a maximum targeting efficiency on tumor cells as well as not being captured by normal cells that consequently lead to a long circulation in blood. In vitro and in vivo studies indicated, in 4 mol% CPP of lipid modified system, that CPP exerted higher efficiency on internalizing the liposomes into targeted subcellular compartments while remaining inactive and free from opsonins at a maximum extent in systemic circulation. The 4% CPPL as a drug delivery system will have great potential in the clinical application of anticancer drugs in future. PMID:26491292

  16. Highly Amino Acid Selective Hydrolysis of Myoglobin at Aspartate Residues as Promoted by Zirconium(IV)-Substituted Polyoxometalates.

    PubMed

    Ly, Hong Giang T; Absillis, Gregory; Janssens, Rik; Proost, Paul; Parac-Vogt, Tatjana N

    2015-06-15

    SDS-PAGE/Edman degradation and HPLC MS/MS showed that zirconium(IV)-substituted Lindqvist-, Keggin-, and Wells-Dawson-type polyoxometalates (POMs) selectively hydrolyze the protein myoglobin at Asp-X peptide bonds under mildly acidic and neutral conditions. This transformation is the first example of highly sequence selective protein hydrolysis by POMs, a novel class of protein-hydrolyzing agents. The selectivity is directed by Asp residues located on the surface of the protein and is further assisted by electrostatic interactions between the negatively charged POMs and positively charged surface patches in the vicinity of the cleavage site. PMID:25950869

  17. Isolation, Purification and Molecular Mechanism of a Peanut Protein-Derived ACE-Inhibitory Peptide

    PubMed Central

    Shi, Aimin; Liu, Hongzhi; Liu, Li; Hu, Hui; Wang, Qiang; Adhikari, Benu

    2014-01-01

    Although a number of bioactive peptides are capable of angiotensin I-converting enzyme (ACE) inhibitory effects, little is known regarding the mechanism of peanut peptides using molecular simulation. The aim of this study was to obtain ACE inhibiting peptide from peanut protein and provide insight on the molecular mechanism of its ACE inhibiting action. Peanut peptides having ACE inhibitory activity were isolated through enzymatic hydrolysis and ultrafiltration. Further chromatographic fractionation was conducted to isolate a more potent peanut peptide and its antihypertensive activity was analyzed through in vitro ACE inhibitory tests and in vivo animal experiments. MALDI-TOF/TOF-MS was used to identify its amino acid sequence. Mechanism of ACE inhibition of P8 was analyzed using molecular docking and molecular dynamics simulation. A peanut peptide (P8) having Lys-Leu-Tyr-Met-Arg-Pro amino acid sequence was obtained which had the highest ACE inhibiting activity of 85.77% (half maximal inhibitory concentration (IC50): 0.0052 mg/ml). This peanut peptide is a competitive inhibitor and show significant short term (12 h) and long term (28 days) antihypertensive activity. Dynamic tests illustrated that P8 can be successfully docked into the active pocket of ACE and can be combined with several amino acid residues. Hydrogen bond, electrostatic bond and Pi-bond were found to be the three main interaction contributing to the structural stability of ACE-peptide complex. In addition, zinc atom could form metal-carboxylic coordination bond with Tyr, Met residues of P8, resulting into its high ACE inhibiting activity. Our finding indicated that the peanut peptide (P8) having a Lys-Leu-Tyr-Met-Arg-Pro amino acid sequence can be a promising candidate for functional foods and prescription drug aimed at control of hypertension. PMID:25347076

  18. Prolyl-specific peptidases for applications in food protein hydrolysis.

    PubMed

    Mika, Nicole; Zorn, Holger; Rühl, Martin

    2015-10-01

    Various food proteins including, e.g. gluten, collagen and casein are rich in L-proline residues. Due to the cyclic structure of proline, these proteins are well protected from enzymatic degradation by typical digestive enzymes. Proline-specific peptidases (PsP) belong to different families of hydrolases acting on peptide bonds (EC 3.4.x.x). They occur in various organisms including bacteria, fungi, plants and insects. Based on their biochemical characteristics, PsP type enzymes are further grouped into different subclasses of which prolyl aminopeptidases (EC 3.4.11.5, PAP), prolyl carboxypeptidases (EC 3.4.17.16, PCP) and prolyl oligopeptidases/prolyl endopeptidases (EC 3.4.21.26, POP/PEP) are of major interest for applications in food biotechnology. This mini review summarises the biochemical assays employed for these subclasses of PsP and their structural properties and the reaction mechanisms. A special focus was set on PsP derived from fungi and insects and important industrial applications in the field of food biotechnology. The degradation of gluten and collagen as well as the hydrolysis of bitter peptides are discussed. PMID:26239067

  19. The hydrolysis of polyimides

    NASA Technical Reports Server (NTRS)

    Hoagland, P. D.; Fox, S. W.

    1973-01-01

    Thermal polymerization of aspartic acid produces a polysuccinimide (I), a chain of aspartoyl residues. An investigation was made of the alkaline hydrolysis of the imide rings of (I) which converts the polyimide to a polypeptide. The alkaline hydrolysis of polyimides can be expected to be kinetically complex due to increasing negative charge generated by carboxylate groups. For this reason, a diimide, phthaloyl-DL-aspartoyl-beta-alanine (IIA) was synthesized for a progressive study of the hydrolysis of polyimides. In addition, this diimide (IIA) can be related to thalidomide and might be expected to exhibit similar reactivity during hydrolysis of the phthalimide ring.

  20. FORMATION OF PEPTIDE BONDS IN SPACE: A COMPREHENSIVE STUDY OF FORMAMIDE AND ACETAMIDE IN Sgr B2(N)

    SciTech Connect

    Halfen, D. T.; Ziurys, L. M.; Ilyushin, V. E-mail: lziurys@as.arizona.edu

    2011-12-10

    acetaldehyde and ketene. CH{sub 3}CONH{sub 2} is therefore one of the most abundant complex organic species in Sgr B2(N), and could be a possible source of larger peptide molecules, as opposed to amino acids.

  1. Hydrolysis of thymic humoral factor gamma 2 by neutral endopeptidase (EC 3.4.24.11).

    PubMed Central

    Indig, F E; Pecht, M; Trainin, N; Burstein, Y; Blumberg, S

    1991-01-01

    A search for the natural substrates for neutral endopeptidase (NEP; EC 3.4.24.11) in the immune system led to investigation of the enzyme's action on thymic humoral factor gamma 2 (THF). The ectoenzyme rapidly and efficiently hydrolyses the Lys6-Phe7 bond of the octapeptide. The site of cleavage was confirmed by h.p.l.c. analysis, amino acid analysis and sequence determination of the products. Phosphoramidon (3.6 microM), a potent inhibitor of the enzyme, prevents this cleavage even during prolonged incubation. The high efficiency of hydrolysis of THF by NEP is similar to that reported for [Leu5]enkephalin, and the dipeptide Phe-Leu is the C-terminal product in the hydrolysis of both peptides. The presence of NEP, reportedly identified as the common acute lymphoblastic leukaemia antigen (CALLA), in bone-marrow cells and other cells of the immune system raises the possibility that it may play a role in modulating the activity of peptides such as THF. PMID:1898375

  2. The major determinant of exendin-4/glucagon-like peptide 1 differential affinity at the rat glucagon-like peptide 1 receptor N-terminal domain is a hydrogen bond from SER-32 of exendin-4*

    PubMed Central

    Mann, RJ; Nasr, NE; Sinfield, JK; Paci, E; Donnelly, D

    2010-01-01

    BACKGROUND AND PURPOSE Exendin-4 (exenatide, Ex4) is a high-affinity peptide agonist at the glucagon-like peptide-1 receptor (GLP-1R), which has been approved as a treatment for type 2 diabetes. Part of the drug/hormone binding site was described in the crystal structures of both GLP-1 and Ex4 bound to the isolated N-terminal domain (NTD) of GLP-1R. However, these structures do not account for the large difference in affinity between GLP-1 and Ex4 at this isolated domain, or for the published role of the C-terminal extension of Ex4. Our aim was to clarify the pharmacology of GLP-1R in the context of these new structural data. EXPERIMENTAL APPROACH The affinities of GLP-1, Ex4 and various analogues were measured at human and rat GLP-1R (hGLP-1R and rGLP-1R, respectively) and various receptor variants. Molecular dynamics coupled with in silico mutagenesis were used to model and interpret the data. KEY RESULTS The membrane-tethered NTD of hGLP-1R displayed similar affinity for GLP-1 and Ex4 in sharp contrast to previous studies using the soluble isolated domain. The selectivity at rGLP-1R for Ex4(9–39) over Ex4(9–30) was due to Ser-32 in the ligand. While this selectivity was not observed at hGLP-1R, it was regained when Glu-68 of hGLP-1R was mutated to Asp. CONCLUSIONS AND IMPLICATIONS GLP-1 and Ex4 bind to the NTD of hGLP-1R with similar affinity. A hydrogen bond between Ser32 of Ex4 and Asp-68 of rGLP-1R, which is not formed with Glu-68 of hGLP-1R, is responsible for the improved affinity of Ex4 at the rat receptor. PMID:20649595

  3. Erythromycin, roxithromycin, and clarithromycin: use of slow-binding kinetics to compare their in vitro interaction with a bacterial ribosomal complex active in peptide bond formation.

    PubMed

    Dinos, George P; Connell, Sean R; Nierhaus, Knud H; Kalpaxis, Dimitrios L

    2003-03-01

    In a cell-free system derived from Escherichia coli, it is shown that clarithromycin and roxithromycin, like their parent compound erythromycin, do not inhibit the puromycin reaction (i.e., the peptide bond formation between puromycin and AcPhe-tRNA bound at the P-site of 70S ribosomes programmed with heteropolymeric mRNA). Nevertheless, all three antibiotics compete for binding on the ribosome with tylosin, a 16-membered ring macrolide that behaves as a slow-binding, slowly reversible inhibitor of peptidyltransferase. The mutually exclusive binding of these macrolides to ribosomes is also corroborated by the fact that they protect overlapping sites in domain V of 23S rRNA from chemical modification by dimethyl sulfate. From this competition effect, detailed kinetic analysis revealed that roxithromycin or clarithromycin (A), like erythromycin, reacts rapidly with AcPhe-tRNA.MF-mRNA x 70S ribosomal complex (C) to form the encounter complex CA which is then slowly isomerized to a more tight complex, termed C*A. The value of the overall dissociation constant, K, encompassing both steps of macrolide interaction with complex C, is 36 nM for erythromycin, 20 nM for roxithromycin, and 8 nM for clarithromycin. Because the off-rate constant of C*A complex does not significantly differ among the three macrolides, the superiority of clarithromycin as an inhibitor of translation in E. coli cells and many Gram-positive bacteria may be correlated with its greater rate of association with ribosomes. PMID:12606769

  4. Progressing batch hydrolysis process

    DOEpatents

    Wright, J.D.

    1985-01-10

    A progressive batch hydrolysis process is disclosed for producing sugar from a lignocellulosic feedstock. It comprises passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with feed stock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feed stock to glucose. The cooled dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, serially fed through a plurality of pre-hydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose. The dilute acid stream containing glucose is cooled after it exits the last prehydrolysis reactor.

  5. Pretreatment and Enzymatic Hydrolysis

    SciTech Connect

    2006-06-01

    Activities in this project are aimed at overcoming barriers associated with high capital and operating costs and sub-optimal sugar yields resulting from pretreatment and subsequent enzymatic hydrolysis of biomass.

  6. Kinetics of the hydrolysis of guanosine 5'-phospho-2-methylimidazolide

    NASA Technical Reports Server (NTRS)

    Kanavarioti, Anastassia

    1986-01-01

    The hydrolysis kinetics of guanosine 5'-phospho-2-methylimidazolide (2-MeImpG) in aqueous buffered solutions of various pH's was studied at 75 and 37 C, using spectrophotometric and HPLC techniques. The hydrolysis was found to be very slow even at low pH. At 75 C and pH at or below l.0, two kinetic processes were observed: the more rapid one was attributed to the hydrolysis of the phosphoimidazolide P-N bond; the second, much slower one, was attributed to the cleavage of the glycosidic bond. It is noted that the P-N hydrolysis in phosphoimidazolides is very slow compared to other phosphoramidates, and that this might be one of the reasons why the phosphoimidazolides showed an extraordinary ability to form long oligomers under template-directed conditions.

  7. Accelerated hydrolysis of substituted cellulose for potential biofuel production: kinetic study and modeling.

    PubMed

    Mu, Bingnan; Xu, Helan; Yang, Yiqi

    2015-11-01

    In this work, kinetics of substitution accelerated cellulose hydrolysis with multiple reaction stages was investigated to lay foundation for mechanism study and molecular design of substituting compounds. High-efficiency hydrolysis of cellulose is critical for cellulose-based bioethanol production. It is known that, substitution could substantially decrease activation energy and increase reaction rate of acidic hydrolysis of glycosidic bonds in cellulose. However, reaction kinetics and mechanism of the accelerated hydrolysis were not fully revealed. In this research, it was proved that substitution therefore accelerated hydrolysis only occurred in amorphous regions of cellulose fibers, and was a process with multiple reaction stages. With molar ratio of substitution less than 1%, the overall hydrolysis rate could be increased for around 10 times. We also quantified the relationship between the hydrolysis rate of individual reaction stage and its major influences, including molar ratio of substitution, activation energy of acidic hydrolysis, pH and temperature. PMID:26253917

  8. Ester-Mediated Amide Bond Formation Driven by Wet-Dry Cycles: A Possible Path to Polypeptides on the Prebiotic Earth.

    PubMed

    Forsythe, Jay G; Yu, Sheng-Sheng; Mamajanov, Irena; Grover, Martha A; Krishnamurthy, Ramanarayanan; Fernández, Facundo M; Hud, Nicholas V

    2015-08-17

    Although it is generally accepted that amino acids were present on the prebiotic Earth, the mechanism by which α-amino acids were condensed into polypeptides before the emergence of enzymes remains unsolved. Here, we demonstrate a prebiotically plausible mechanism for peptide (amide) bond formation that is enabled by α-hydroxy acids, which were likely present along with amino acids on the early Earth. Together, α-hydroxy acids and α-amino acids form depsipeptides-oligomers with a combination of ester and amide linkages-in model prebiotic reactions that are driven by wet-cool/dry-hot cycles. Through a combination of ester-amide bond exchange and ester bond hydrolysis, depsipeptides are enriched with amino acids over time. These results support a long-standing hypothesis that peptides might have arisen from ester-based precursors. PMID:26201989

  9. Ester-Mediated Amide Bond Formation Driven by Wet–Dry Cycles: A Possible Path to Polypeptides on the Prebiotic Earth**

    PubMed Central

    Forsythe, Jay G; Yu, Sheng-Sheng; Mamajanov, Irena; Grover, Martha A; Krishnamurthy, Ramanarayanan; Fernández, Facundo M; Hud, Nicholas V

    2015-01-01

    Although it is generally accepted that amino acids were present on the prebiotic Earth, the mechanism by which α-amino acids were condensed into polypeptides before the emergence of enzymes remains unsolved. Here, we demonstrate a prebiotically plausible mechanism for peptide (amide) bond formation that is enabled by α-hydroxy acids, which were likely present along with amino acids on the early Earth. Together, α-hydroxy acids and α-amino acids form depsipeptides—oligomers with a combination of ester and amide linkages—in model prebiotic reactions that are driven by wet–cool/dry–hot cycles. Through a combination of ester–amide bond exchange and ester bond hydrolysis, depsipeptides are enriched with amino acids over time. These results support a long-standing hypothesis that peptides might have arisen from ester-based precursors. PMID:26201989

  10. Progressing batch hydrolysis process

    DOEpatents

    Wright, John D.

    1986-01-01

    A progressive batch hydrolysis process for producing sugar from a lignocellulosic feedstock, comprising passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feedstock to glucose; cooling said dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, then feeding said dilute acid stream serially through a plurality of prehydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose; and cooling the dilute acid stream containing glucose after it exits the last prehydrolysis reactor.

  11. Acid hydrolysis of cellulose

    SciTech Connect

    Salazar, H.

    1980-12-01

    One of the alternatives to increase world production of etha nol is by the hydrolysis of cellulose content of agricultural residues. Studies have been made on the types of hydrolysis: enzimatic and acid. Data obtained from the sulphuric acid hydrolysis of cellulose showed that this process proceed in two steps, with a yield of approximately 95% glucose. Because of increases in cost of alternatives resources, the high demand of the product and the more economic production of ethanol from cellulose materials, it is certain that this technology will be implemented in the future. At the same time further studies on the disposal and reuse of the by-products of this production must be undertaken.

  12. Reaction Dynamics of ATP Hydrolysis Catalyzed by P-Glycoprotein

    PubMed Central

    2015-01-01

    P-glycoprotein (P-gp) is a member of the ABC transporter family that confers drug resistance to many tumors by catalyzing their efflux, and it is a major component of drug–drug interactions. P-gp couples drug efflux with ATP hydrolysis by coordinating conformational changes in the drug binding sites with the hydrolysis of ATP and release of ADP. To understand the relative rates of the chemical step for hydrolysis and the conformational changes that follow it, we exploited isotope exchange methods to determine the extent to which the ATP hydrolysis step is reversible. With γ18O4-labeled ATP, no positional isotope exchange is detectable at the bridging β-phosphorus–O−γ-phosphorus bond. Furthermore, the phosphate derived from hydrolysis includes a constant ratio of three 18O/two 18O/one 18O that reflects the isotopic composition of the starting ATP in multiple experiments. Thus, H2O-exchange with HPO42– (Pi) was negligible, suggesting that a [P-gp·ADP·Pi] is not long-lived. This further demonstrates that the hydrolysis is essentially irreversible in the active site. These mechanistic details of ATP hydrolysis are consistent with a very fast conformational change immediately following, or concomitant with, hydrolysis of the γ-phosphate linkage that ensures a high commitment to catalysis in both drug-free and drug-bound states. PMID:24506763

  13. Inhibitory activity of thermal copolymers of amino acids for the metal-catalyzed hydrolysis of an RNA dinucleotide

    NASA Astrophysics Data System (ADS)

    Kawamura, Kunio; Nagahama, Minoru; Yao, Toshio

    2006-01-01

    It is well known that the hydrolysis of RNA is substantially catalyzed by several metal ions. Although this fact poses a problem for the RNA world hypothesis, there may have been unknown pathways for the protection of RNA molecules against the hydrolytic degradation under the primitive earth conditions. Thus, we have investigated whether or not thermal copolymers of amino acids (TCAA) inhibit the catalytic activity of metal ions for the RNA hydrolyses; TCAA is a suitable model material for prebiotic protein-like molecules since TCAA involving peptide bonding is readily prepared by heating amino acid mixtures under prebiotic conditions. The activities of metal ions that Fe(III) and Co(II) enhance somewhat the 3',5'-cytidylylguanosine (CpG) hydrolysis and Ce(III) and Eu(III) accelerate greatly the CpG hydrolysis were notably reduced by TCAA. This fact indicates that protein-like molecules would have played important roles for the accumulation of RNA under the primitive earth conditions.

  14. Vibrational analysis of amino acids and short peptides in hydrated media. 3. Successive KL repeats induce highly stable beta-strands capable of forming non-H-bonded aggregates.

    PubMed

    Guiffo-Soh, Guy; Hernandez, Belén; Coïc, Yves-Marie; Boukhalfa-Heniche, Fatima-Zohra; Fadda, Giulia; Ghomi, Mahmoud

    2008-01-31

    Circular dichroism (CD) and Raman scattering were applied to the aqueous solution of minimalist LK peptides constructed with successive KL repeats leading to the following generic primary sequence: (KL)nK. Three peptides of this family, a 3-mer (n=1), a 9-mer (n=4), and a 15-mer (n=7), are analyzed in this report. Raman spectra of the 3-mer (KLK, a random chain) and its labile-hydrogen deuterated species yield a set of interesting information for analyzing longer peptides of this series. Although the CD spectrum of the 9-mer (KLKLKLKLK) reveals a signal traditionally assigned to a random structure, the corresponding Raman spectrum allows finding a mixture of conformations in solution, adopting predominantly beta-type structures. This fact proves the utility of Raman spectroscopy to eliminate eventual ambiguity concerning conformational assignments in peptides based only on the use of CD technique. Finally, the 15-mer (KLKLKLKLKLKLKLK) gives rise to CD and Raman spectra clearly assignable to a beta-type structure. On the basis of all the observed results on the 15-mer, we can confirm that this peptide may exist as isolated beta-strands at low concentration (sub-micromolar), flat-oriented at the air/water interface, whereas at high concentrations (millimolar), non-H-bonded immersible aggregates might be formed. A hypothetical model for these beta-strand aggregates could be proposed as stabilized by an interior hydrophobic core and a hydrophilic external face, formed by leucine and lysine side chains, respectively. PMID:18177032

  15. Theoretical investigation of the role of clay edges in prebiotic peptide bond formation. II - Structures and thermodynamics of the activated complex species

    NASA Technical Reports Server (NTRS)

    Collins, Jack R.; Loew, Gilda H.; Luke, Brian T.; White, David H.

    1988-01-01

    Molecular orbital calculations are used to study amino acid activation by anhydride formation in neutral phosphates and in tetrahedral silicate and aluminate sites on clay edges. The results agree with previous ab initio studies of Luke et al. (1984) on the reactant species. Relative heats of formation of the anhydrides indicate the extent of anhydride formation to be the greatest for Al and the least for phosphate, which is the same order as the stability of hydrolysis.

  16. Fluorine Scan of Inhibitors of the Cysteine Protease Human Cathepsin L: Dipolar and Quadrupolar Effects in the π-Stacking of Fluorinated Phenyl Rings on Peptide Amide Bonds.

    PubMed

    Giroud, Maude; Harder, Michael; Kuhn, Bernd; Haap, Wolfgang; Trapp, Nils; Schweizer, W Bernd; Schirmeister, Tanja; Diederich, François

    2016-05-19

    The π-stacking of fluorinated benzene rings on protein backbone amide groups was investigated, using a dual approach comprising enzyme-ligand binding studies complemented by high-level quantum chemical calculations. In the experimental study, the phenyl substituent of triazine nitrile inhibitors of human cathepsin L (hCatL), which stacks onto the peptide amide bond Gly67-Gly68 at the entrance of the S3 pocket, was systematically fluorinated, and differences in inhibitory potency were measured in a fluorimetric assay. Binding affinity is influenced by lipophilicity (clog P), the dipole and quadrupole moments of the fluorinated rings, but also by additional interactions of the introduced fluorine atoms with the local environment of the pocket. Generally, the higher the degree of fluorination, the better the binding affinities. Gas phase calculations strongly support the contributions of the molecular quadrupole moments of the fluorinated phenyl rings to the π-stacking interaction with the peptide bond. These findings provide useful guidelines for enhancing π-stacking on protein amide fragments. PMID:27095165

  17. Exothermic Bond Breaking: A Persistent Misconception

    ERIC Educational Resources Information Center

    Galley, William C.

    2004-01-01

    The misconceptions regarding the nature of ATP hydrolysis and bond breaking are discussed. The students' knowledge in this area is quantitatively measured by a survey of over 600 biochemistry and physiology students.

  18. Effect of degree of hydrolysis of whey protein on in vivo plasma amino acid appearance in humans.

    PubMed

    Farup, Jean; Rahbek, Stine Klejs; Storm, Adam C; Klitgaard, Søren; Jørgensen, Henry; Bibby, Bo M; Serena, Anja; Vissing, Kristian

    2016-01-01

    Whey protein is generally found to be faster digested and to promote faster and higher increases in plasma amino acid concentrations during the immediate ~60 min following protein ingestion compared to casein. The aim of the present study was to compare three different whey protein hydrolysates with varying degrees of hydrolysis (DH, % cleaved peptide bonds) to evaluate if the degree of whey protein hydrolysis influences the rate of amino acid plasma appearance in humans. A casein protein was included as reference. The three differentially hydrolysed whey proteins investigated were: High degree of hydrolysis (DH, DH = 48 %), Medium DH (DH = 27 %), and Low DH (DH = 23 %). The casein protein was intact. Additionally, since manufacturing of protein products may render some amino acids unavailable for utilisation in the body the digestibility and the biological value of all four protein fractions were evaluated in a rat study. A two-compartment model for the description of the postprandial plasma amino acid kinetics was applied to investigate the rate of postprandial total amino acid plasma appearance of the four protein products. The plasma amino acid appearance rates of the three whey protein hydrolysates (WPH) were all significantly higher than for the casein protein, however, the degree of hydrolysis of the WPH products did not influence plasma total amino acid appearance rate (estimates of DH and 95 % confidence intervals [CI] (mol L(-1) min(-1)): High DH 0.0585 [0.0454, 0.0754], Medium DH 0.0594 [0.0495, 0.0768], Low DH 0.0560 [0.0429, 0.0732], Casein 0.0194 [0.0129, 0.0291]). The four protein products were all highly digestible, while the biological value decreased with increasing degree of hydrolysis. In conclusion, the current study does not provide evidence that the degree of whey protein hydrolysis is a strong determinant for plasma amino acid appearance rate within the studied range of hydrolysis and protein dose. PMID:27065230

  19. Breaking the low barrier hydrogen bond in a serine protease.

    PubMed Central

    Kidd, R. D.; Sears, P.; Huang, D. H.; Witte, K.; Wong, C. H.; Farber, G. K.

    1999-01-01

    The serine protease subtilisin BPN' is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water-miscible organic co-solvent such as N,N-dimethylformamide (DMF). However, in 50% DMF, the k(cat) for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the k(cat) for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397+1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 A resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 180 degrees around the Cbeta-Cgamma bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co-solvents. PMID:10048334

  20. Influence of the rapeseed protein hydrolysis process on CHO cell growth.

    PubMed

    Chabanon, G; Alves da Costa, L; Farges, B; Harscoat, C; Chenu, S; Goergen, J-L; Marc, A; Marc, I; Chevalot, I

    2008-10-01

    Different protein hydrolysates were prepared from enzymatic hydrolyses of a rapeseed isolate (>90% protein content) using different commercial enzymes of non-animal origin. The extent of hydrolysis was controlled to produce hydrolysates corresponding to various degrees of hydrolysis (DH) from 5 to 30. These hydrolysates were characterized according to their solubility and size peptide pattern. Different growth behaviours of Chinese Hamster Ovary cells were observed when these various hydrolysates were added in serum-free medium containing transferrin, albumin and insulin. Hydrolysates from low degree of hydrolysis generally did not exhibit significant positive effect on cell growth; conversely hydrolysates from extensive hydrolysis, corresponding to a major low molecular size peptides content, usually allowed an increase of the maximal cell density. However, depending on the enzyme used, the supplementation with hydrolysates corresponding to a high degree of hydrolysis and composed of at least 70% peptides with a molecular size under 1kDa, led to different maximal cell density values, indicating the importance of enzyme specificity and consequently the nature of the released peptides. This result showed that the positive influence of the rapeseed hydrolysates on cell growth was not only due to a nutritional support tied to the addition of small peptides but may be related to the presence of peptides exhibiting growth or survival factor effects. Furthermore, total substitution of proteins (transferrin, albumin and insulin) in the cell culture medium by some rapeseed hydrolysates appeared to be a promising alternative to improve the cell growth in protein-free media. PMID:18296044

  1. Site-Specific Hydrolysis Reaction C-Terminal of Methionine in Met-His during Metal-Catalyzed Oxidation of IgG-1.

    PubMed

    Mozziconacci, Olivier; Arora, Jayant; Toth, Ronald T; Joshi, Sangeeta B; Zhou, Shuxia; Volkin, David B; Schöneich, Christian

    2016-04-01

    The metal-catalyzed oxidation by [Fe(II)(EDTA)](2-)/H2O2 of IgG-1 leads to the site-specific hydrolysis of peptide bonds in the Fc region. The major hydrolytic cleavage occurs between Met428 and His429, consistent with a mechanism reported for the site-specific hydrolysis of parathyroid hormone (1-34) between Met8 and His9 (Mozziconacci, O.; et al. Mol. Pharmaceutics 2013, 10 (2), 739-755). In IgG-1, to a lesser extent, we also observe hydrolysis reactions between Met252 and Ile253. After 2 h of oxidation (at pH 5.8, 37 °C) approximately 5% of the protein is cleaved between Met428 and His429. For comparison, after 2 h of oxidation, the amount of tryptic peptides containing a Met sulfoxide residue represents less than 0.1% of the protein. The effect of this site-specific hydrolysis on the conformational stability and aggregation propensity of the antibody was also examined. No noticeable differences in structural integrity and conformational stability were observed between control and oxidized IgG-1 samples as measured by circular dichroism (CD), fluorescence spectroscopy, and static light scattering (SLS). Small amounts of soluble and insoluble aggregates (3-6%) were, however, observed in the oxidized samples by UV-visible absorbance spectroscopy and size exclusion chromatography (SEC). Over the course of metal-catalyzed oxidation, increasing amounts of fragments were also observed by SEC. An increase in the concentration of subvisible particles was detected by microflow imaging (MFI). PMID:26942274

  2. Slow step after bond-breaking by porcine pepsin identified using solvent deuterium isotope effects

    SciTech Connect

    Rebholz, K.L.; Northrop, D.B. )

    1991-04-15

    The relatively fast artificial substrate Leu-Ser-rho-nitro-Phe-Nle-Ala-Leu-OMe generates a solvent isotope effect of 1.51 +/- 0.02 only on the maximal velocity of peptide hydrolysis catalyzed by porcine pepsin. The absence of an isotope effect on V/K places the isotopically-sensitive step after peptide bond cleavage and the release of the first product. Reprotonation of the active site aspartic carboxyls is proposed as the most likely interpretation of this observation. Structural and kinetic similarities between pepsin and other aspartic proteinases, including the therapeutically important targets HIV protease and renin, suggest a similar slow reprotonation step after catalysis. This mechanistic feature has important implications regarding inhibitor design; if most of the enzymes are present in a product-release form during steady-state turnover, then perhaps inhibitors should be designed as product analogs instead of substrate analogs.

  3. Discovery and Mechanistic Studies of Facile N-Terminal Cα–C Bond Cleavages in the Dissociation of Tyrosine-Containing Peptide Radical Cations

    SciTech Connect

    Mu, Xiaoyan; Song, Tao; Xu, Minjie; Lai, Cheuk-Kuen; Siu, Chi-Kit; Laskin, Julia; Chu, Ivan K.

    2014-03-28

    Gas phase fragmentations of protein and peptide (M) ions in a mass spectrometer—induced by, for example, electron-capture dissociation1-2 and electron-transfer dissociation3-422 —form the foundation for top-down amino acid sequencing approaches for the rapid identification of protein components in complex biological samples. During these processes, protonated protein and peptide radicals ([M + nH]•(n – 1)+)5–8 are generated; their fragmentations are governed largely by the properties of the unpaired electron. Because of their importance in modern bioanalytical chemistry, considerable attention has been drawn recently toward understanding the radical cation chemistry behind the fragmentations of these odd-electron biomolecular ions in the gas phase.

  4. The structure of salt bridges between Arg+ and Glu- in peptides investigated with 2D-IR spectroscopy: Evidence for two distinct hydrogen-bond geometries

    NASA Astrophysics Data System (ADS)

    Huerta-Viga, Adriana; Amirjalayer, Saeed; Domingos, Sérgio R.; Meuzelaar, Heleen; Rupenyan, Alisa; Woutersen, Sander

    2015-06-01

    Salt bridges play an important role in protein folding and in supramolecular chemistry, but they are difficult to detect and characterize in solution. Here, we investigate salt bridges between glutamate (Glu-) and arginine (Arg+) using two-dimensional infrared (2D-IR) spectroscopy. The 2D-IR spectrum of a salt-bridged dimer shows cross peaks between the vibrational modes of Glu- and Arg+, which provide a sensitive structural probe of Glu-⋯Arg+ salt bridges. We use this probe to investigate a β-turn locked by a salt bridge, an α-helical peptide whose structure is stabilized by salt bridges, and a coiled coil that is stabilized by intra- and intermolecular salt bridges. We detect a bidentate salt bridge in the β-turn, a monodentate one in the α-helical peptide, and both salt-bridge geometries in the coiled coil. To our knowledge, this is the first time 2D-IR has been used to probe tertiary side chain interactions in peptides, and our results show that 2D-IR spectroscopy is a powerful method for investigating salt bridges in solution.

  5. Evaluation of the Ser-His Dipeptide, a Putative Catalyst of Amide and Ester Hydrolysis.

    PubMed

    MacDonald, Melissa J; Lavis, Luke D; Hilvert, Donald; Gellman, Samuel H

    2016-08-01

    Efficient hydrolysis of amide bonds has long been a reaction of interest for organic chemists. The rate constants of proteases are unmatched by those of any synthetic catalyst. It has been proposed that a dipeptide containing serine and histidine is an effective catalyst of amide hydrolysis, based on an apparent ability to degrade a protein. The capacity of the Ser-His dipeptide to catalyze the hydrolysis of several discrete ester and amide substrates is investigated using previously described conditions. This dipeptide does not catalyze the hydrolysis of amide or unactivated ester groups in any of the substrates under the conditions evaluated. PMID:27400366

  6. Antimitotic peptides and depsipeptides.

    PubMed

    Hamel, Ernest; Covell, David G

    2002-01-01

    Tubulin is the target for an ever increasing number of unusual peptides and depsipeptides that were originally isolated from a wide variety of organisms. Since tubulin is the major component of cellular microtubules, which maintain cell shape in interphase and form the mitotic spindle, most of these compounds are highly toxic to mammalian cells. These peptides and depsipeptides disrupt cellular microtubules and prevent formation of a functional spindle, resulting in the accumulation of cultured cells in the G2/M phase of the cell cycle through specific inhibition of mitosis. At the biochemical level, the compounds all inhibit the assembly of tubulin into polymer and, in the cases where it has been studied, strongly suppress microtubule dynamics at low concentrations. In most cases the peptides and depsipeptides inhibit the binding of vinblastine and vincristine to tubulin in a noncompetitive manner, inhibit tubulin-dependent GTP hydrolysis, and interfere with nucleotide turnover at the exchangeable GTP site on beta-tubulin. Most of the peptides and depsipeptides induce tubulin to form oligomers of aberrant morphology, including tubulin rings that vary in diameter depending on the (depsi) peptide under study. The purpose of this review is to give an overview of the cellular, biochemical, in vivo, and SAR aspects of this group of compounds. We also summarize initial efforts by computer modeling to decipher a pharmacophore among the diverse structures of these peptides and depsipeptides. PMID:12678750

  7. Hydrolysis of biomass material

    DOEpatents

    Schmidt, Andrew J.; Orth, Rick J.; Franz, James A.; Alnajjar, Mikhail

    2004-02-17

    A method for selective hydrolysis of the hemicellulose component of a biomass material. The selective hydrolysis produces water-soluble small molecules, particularly monosaccharides. One embodiment includes solubilizing at least a portion of the hemicellulose and subsequently hydrolyzing the solubilized hemicellulose to produce at least one monosaccharide. A second embodiment includes solubilizing at least a portion of the hemicellulose and subsequently enzymatically hydrolyzing the solubilized hemicellulose to produce at least one monosaccharide. A third embodiment includes solubilizing at least a portion of the hemicellulose by heating the biomass material to greater than 110.degree. C. resulting in an aqueous portion that includes the solubilized hemicellulose and a water insoluble solids portion and subsequently separating the aqueous portion from the water insoluble solids portion. A fourth embodiment is a method for making a composition that includes cellulose, at least one protein and less than about 30 weight % hemicellulose, the method including solubilizing at least a portion of hemicellulose present in a biomass material that also includes cellulose and at least one protein and subsequently separating the solubilized hemicellulose from the cellulose and at least one protein.

  8. Hydrolysis of cisplatin--a first-principles metadynamics study.

    PubMed

    Lau, Justin Kai-Chi; Ensing, Bernd

    2010-09-21

    Cisplatin, or cis-[Pt(NH(3))(2)Cl(2)], was the first member of a new revolutionary class of anticancer drugs that is still used today for the treatment of a wide variety of cancers. The mode of action of cisplatin starts inside the cell with the hydrolysis of Pt-Cl bonds to form a Pt-aqua complex. The solvent environment plays an essential role in many biochemical processes in general, and is expected to have a particular strong effect on the activation (hydrolysis) of cisplatin and cisplatin derivatives. To investigate these solvent effects, we have studied the explicit solvent structures during cisplatin hydrolysis by means of Car-Parrinello molecular dynamics simulations. Since hydrolysis is an activated process, and thus a rare event on the simulation timescale, we have applied the metadynamics sampling technique to map out the free energy landscape from which the reaction mechanism and activation free energy are obtained. Our simulations show that hydrogen bonding between solvent water molecules and metal complexes in the hydrolyzed product systems is stronger than that in the reactant cisplatin system. In addition, the free energy profiles from our metadynamics simulations for the cisplatin hydrolysis shows that the second hydrolysis of cisplatin is thermodynamically favourable, which is in good agreement with experimental results and previous static density functional theory calculations. The reactant channels for both hydrolysis steps are rather wide and flat, indicative of a continuous spectrum of allowed mechanisms with no strong preference for either concerted dissociative or concerted associative pathways. Three or five coordinated metastable intermediates do not exist in aqueous solution. PMID:20582358

  9. Collagencin, an antibacterial peptide from fish collagen: Activity, structure and interaction dynamics with membrane.

    PubMed

    Ennaas, Nadia; Hammami, Riadh; Gomaa, Ahmed; Bédard, François; Biron, Éric; Subirade, Muriel; Beaulieu, Lucie; Fliss, Ismail

    2016-04-29

    In this study, we first report characterization of collagencin, an antimicrobial peptide identified from fish collagen hydrolysate. The peptide completely inhibited the growth of Staphylococcus aureus at 1.88 mM. Although non-toxic up to 470 μM, collagencin was hemolytic at higher concentrations. The secondary structure of collagencin was mainly composed by β-sheet and β-turn as determined by CD measurements and molecular dynamics. The peptide is likely to form β-sheet structure under hydrophobic environments and interacts with both anionic (phosphatidylglycerol) and zwitterionic (phosphoethanolamine and phosphatidylcholine) lipids as shown with CD spectroscopy and molecular dynamics. The peptide formed several hydrogen bonds with both POPG and POPE lipids and remained at membrane-water interface, suggesting that collagencin antibacterial action follows a carpet mechanism. Collagenous fish wastes could be processed by enzymatic hydrolysis and transformed into products of high value having functional or biological properties. Marine collagens are a promising source of antimicrobial peptides with new implications in food safety and human health. PMID:27038545

  10. Preparation and antihypertensive activity of peptides from Porphyra yezoensis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This research was to develop an antihypertensive peptide, an efficient angiotensin converting enzyme (ACE) inhibitor (ACEI), from Porphyra yezoensis. Seven commercial enzymes were screened and then enzymatic hydrolysis conditions were optimised. The results showed that alcalase was the most effectiv...

  11. The Mechanisms of Plant Cell Wall Deconstruction during Enzymatic Hydrolysis

    PubMed Central

    Thygesen, Lisbeth G.; Thybring, Emil E.; Johansen, Katja S.; Felby, Claus

    2014-01-01

    Mechanical agitation during enzymatic hydrolysis of insoluble plant biomass at high dry matter contents is indispensable for the initial liquefaction step in biorefining. It is known that particle size reduction is an important part of liquefaction, but the mechanisms involved are poorly understood. Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry, particularly when it comes to up-scaling of processes based on insoluble feed stocks. PMID:25232741

  12. Migration and transformation rule of heavy metals in sludge during hydrolysis for protein extraction.

    PubMed

    Li, Yulong; Xue, Fei; Li, Jiebing; Xu, Shi Hong; Li, Dengxin

    2016-03-01

    The content and speciation of heavy metals can fundamentally affect the hydrolysis of sludge. This research study investigates the migration and transformation rule of heavy metals during the hydrolysis process by measuring the content of exchangeables (F1), bound to carbonates (F2), bound to Fe-Mn oxides (F3), bound to organic matter (F4), and residuals (F5) under different periods of time undergoing hydrolysis. The results show that the hydrolysis process generally stabilized Cu, Zn, Mn, Ni, Pb, Cr, and As by transforming the unstable states into structurally stable states. Such transformations and stabilization were primarily caused by the changes in local metal ion environment and bonding structure, oxidation of sulfides, pyrolyzation of organic matter, and evaporation of resulting volatile materials. An X-ray diffractometry (XRD) of the residuals conducted after hydrolysis indicated that hydrolysis did have a significant influence on the transportation and transformation of heavy metals. PMID:26564189

  13. Determination of angiotensin I-converting enzyme activity in cell culture using fluorescence resonance energy transfer peptides.

    PubMed

    Sabatini, R A; Bersanetti, P A; Farias, S L; Juliano, L; Juliano, M A; Casarini, D E; Carmona, A K; Paiva, A C M; Pesquero, J B

    2007-04-15

    An assay using fluorescence resonance energy transfer peptides was developed to assess angiotensin I-converting enzyme (ACE) activity directly on the membrane of transfected Chinese hamster ovary cells (CHO) stably expressing the full-length somatic form of the enzyme. The advantage of the new method is the possibility of using selective substrates for the two active sites of the enzyme, namely Abz-FRK(Dnp)P-OH for somatic ACE, Abz-SDK(Dnp)P-OH for the N domain, and Abz-LFK(Dnp)-OH for the C domain. Hydrolysis of a peptide bond between the donor/acceptor pair (Abz/Dnp) generates detectable fluorescence, allowing quantitative measurement of the enzymatic activity. The kinetic parameter K(m) for the hydrolysis of the three substrates by ACE in this system was also determined and the values are comparable to those obtained using the purified enzyme in solution. The specificity of the activity was demonstrated by the complete inhibition of the hydrolysis by the ACE inhibitor lisinopril. Therefore, the results presented in this work show for the first time that determination of ACE activity directly on the surface of intact CHO cells is feasible and that the method is reliable and sensitive. In conclusion, we describe a methodology that may represent a new tool for the assessment of ACE activity which will open the possibility to study protein interactions in cells in culture. PMID:17320031

  14. An optimal hydrogen-bond surrogate for α-helices.

    PubMed

    Joy, Stephen T; Arora, Paramjit S

    2016-04-14

    Substitution of a main chain i → i + 4 hydrogen bond with a covalent bond can nucleate and stabilize the α-helical conformation in peptides. Herein we describe the potential of different alkene isosteres to mimic intramolecular hydrogen bonds and stabilize α-helices in diverse peptide sequences. PMID:27046675

  15. Comparing the catalytic strategy of ATP hydrolysis in biomolecular motors.

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2016-07-27

    ATP-driven biomolecular motors utilize the chemical energy obtained from the ATP hydrolysis to perform vital tasks in living cells. Understanding the mechanism of enzyme-catalyzed ATP hydrolysis reaction has substantially progressed lately thanks to combined quantum/classical molecular mechanics (QM/MM) simulations. Here, we present a comparative summary of the most recent QM/MM results for myosin, kinesin and F1-ATPase motors. These completely different motors achieve the acceleration of ATP hydrolysis through a very similar catalytic mechanism. ATP hydrolysis has high activation energy because it involves the breaking of two strong bonds, namely the Pγ-Oβγ bond of ATP and the H-O bond of lytic water. The key to the four-fold decrease in the activation barrier by the three enzymes is that the breaking of the Pγ-Oβγ bond precedes the deprotonation of the lytic water molecule, generating a metaphosphate hydrate complex. The resulting singly charged trigonal planar PγO3(-) metaphosphate is a better electrophilic target for attack by an OaH(-) hydroxyl group. The formation of this OaH(-) is promoted by a strong polarization of the lytic water: in all three proteins, this water is forming a hydrogen-bond with a backbone carbonyl group and interacts with the carboxylate group of glutamate (either directly or via an intercalated water molecule). This favors the shedding of one proton by the attacking water. The abstracted proton is transferred to the γ-phosphate via various proton wires, resulting in a H2PγO4(-)/ADP(3-) product state. This catalytic strategy is so effective that most other nucleotide hydrolyzing enzymes adopt a similar approach, as suggested by their very similar triphosphate binding sites. PMID:27296627

  16. Bent Bonds and Multiple Bonds.

    ERIC Educational Resources Information Center

    Robinson, Edward A.; Gillespie, Ronald J.

    1980-01-01

    Considers carbon-carbon multiple bonds in terms of Pauling's bent bond model, which allows direct calculation of double and triple bonds from the length of a CC single bond. Lengths of these multiple bonds are estimated from direct measurements on "bent-bond" models constructed of plastic tubing and standard kits. (CS)

  17. ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS. I. ALKALINE HYDROLYSIS

    EPA Science Inventory

    SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to allow the calculation of alkaline hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition state...

  18. ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS - ALKALINE HYDROLYSIS

    EPA Science Inventory

    SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to allow the calculation of alkaline hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition state...

  19. Substrate specificity of the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) assessed by mutagenesis and analysis of synthetic peptides: substrate residues distant from the scissile bond are critical for proteolysis.

    PubMed Central

    Laursen, Lisbeth S; Overgaard, Michael T; Nielsen, Claus G; Boldt, Henning B; Hopmann, Kathrin H; Conover, Cheryl A; Sottrup-Jensen, Lars; Giudice, Linda C; Oxvig, Claus

    2002-01-01

    Human pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor (IGF) binding protein-4 (IGFBP-4), causing a dramatic reduction in its affinity for IGF-I and -II. Through this mechanism, PAPP-A is a regulator of IGF bioactivity in several systems, including the human ovary and the cardiovascular system. PAPP-A belongs to the metzincin superfamily of zinc metalloproteinases, and is the founding member of a fifth metzincin family, the pappalysins. Herein, we first determined that PAPP-A cleaves IGFBP-4 at a single site (Met-135/Lys-136), and we analysed the influence of ionic strength, pH and zinc ion concentration on the cleavage reaction. Secondly, we sought to delineate the role of substrate residues in PAPP-A-mediated cleavage by the construction and analysis of 30 IGFBP-4 mutants in which various residues were replaced by alanine, by the analysis of eight mutants of IGFBP-5 (found recently to be a second PAPP-A substrate), and by cleavage analysis of synthetic peptides derived from IGFBP-4. Our data reveal a complex mode of substrate recognition and/or binding, pointing at important roles for several basic residues located up to 16 residues N-terminal to the scissile bond. An unexpected parallel can be drawn with an intracellular enzyme, the mitochondrial processing peptidase, that may help us to understand properties of the pappalysins. Further, proteinase-resistant variants of IGFBP-4 and -5, presented here, will be useful tools for the study of proteolysis in cell-based systems, and our finding that a synthetic peptide can be cleaved by PAPP-A provides the basis for development of quantitative assays for the investigation of PAPP-A enzyme kinetics. PMID:12241545

  20. Functional coupling of Cys-226 and Cys-296 in the glucagon-like peptide-1 (GLP-1) receptor indicates a disulfide bond that is close to the activation pocket.

    PubMed

    Mann, Rosalind J; Al-Sabah, Suleiman; de Maturana, Rakel López; Sinfield, John K; Donnelly, Dan

    2010-12-01

    G protein-coupled receptors (GPCRs) are seven transmembrane α-helical (7TM) integral membrane proteins that play a central role in both cell signaling and in the action of many pharmaceuticals. The crystal structures of several Family A GPCRs have shown the presence of a disulfide bond linking transmembrane helix 3 (TM3) to the second extracellular loop (ECL2), enabling ECL2 to stabilize and contribute to the ligand binding pocket. Family B GPCRs share no significant sequence identity with those in Family A but nevertheless share two conserved cysteines in topologically equivalent positions. Since there are no available crystal structures for the 7TM domain of any Family B GPCR, we used mutagenesis alongside pharmacological analysis to investigate the role of ECL2 and the conserved cysteine residues. We mutated Cys-226, at the extracellular end of TM3 of the glucagon-like peptide-1 (GLP-1) receptor, to alanine and observed a 38-fold reduction in GLP-1 potency. Interestingly, this potency loss was restored by the additional substitution of Cys-296 in ECL2 to alanine. Alongside the complete conservation of these cysteine residues in Family B GPCRs, this functional coupling suggested the presence of a disulfide bond. Further mutagenesis demonstrated that the low potency observed at the C226A mutant, compared with the C226A-C296A double mutant, was the result of the bulky nature of the released Cys-296 side chain. Since this suggested that ECL2 was in close proximity to the agonist activation pocket, an alanine scan of ECL2 was carried out which confirmed the important role of this loop in agonist-induced receptor activation. PMID:20869417

  1. Dry heat forced degradation of buserelin peptide: kinetics and degradant profiling.

    PubMed

    D'Hondt, Matthias; Fedorova, Maria; Peng, Chien-Yu; Gevaert, Bert; Taevernier, Lien; Hoffmann, Ralf; De Spiegeleer, Bart

    2014-06-01

    Buserelin is a GnRH agonist peptide drug, comprising a nine amino acid sequence (pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NH-Et) and most commonly known for its application in hormone dependent cancer therapy, e.g. prostate cancer. In order to evaluate its hot-melt extrusion (HME) capabilities, buserelin powder in its solid state was exposed to elevated temperatures for prolonged time periods. A stability indicating UPLC-PDA method was used for quantification of buserelin and the formed degradants. Different solid state kinetic models were statistically evaluated of which the Ginstling-Brounshtein model fitted the data best. Extrapolation to and experimental verification of typical HME-related conditions, i.e. 5 min at 100°C and 125°C, showed no significant degradation, thus demonstrating the HME capabilities of buserelin. Mass spectrometric identification of the buserelin-related degradants formed under solid state heat stress was performed. Based upon the identity of these degradants, different degradation hypotheses were raised. First, direct β-elimination of the hydroxyl moiety at the serine residue, followed by fragmentation into an amide (pGlu-His-Trp-NH2) and pyruvoyl (pyruvoyl-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NH-Et) peptide fragments, was postulated. Alternatively, internal esterification due to nucleophilic attack of the unprotected serine residue, followed by β-elimination or hydrolysis would yield pGlu-His-Trp, pGlu-His-Trp-NH2 and the pyruvoyl peptide fragment. Degradant pGlu-His-Trp-Ser-Tyr-NH2 is believed to be formed in a similar way. Secondly, direct backbone hydrolysis would yield pGlu-His-Trp and Tyr-D-Ser(tBu)-Leu-Arg-Pro-NH-Et peptide fragments. Moreover, the presence of Ala-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NH-Et can be explained by hydrolysis of the Trp-Ser peptide bond and conversion of the serine moiety to an alanine moiety. Third and finally, isomerisation of aforementioned peptide fragments and buserelin itself was also observed. PMID:24657556

  2. Hydrolysis reactor for hydrogen production

    SciTech Connect

    Davis, Thomas A.; Matthews, Michael A.

    2012-12-04

    In accordance with certain embodiments of the present disclosure, a method for hydrolysis of a chemical hydride is provided. The method includes adding a chemical hydride to a reaction chamber and exposing the chemical hydride in the reaction chamber to a temperature of at least about 100.degree. C. in the presence of water and in the absence of an acid or a heterogeneous catalyst, wherein the chemical hydride undergoes hydrolysis to form hydrogen gas and a byproduct material.

  3. Kinetics and mechanism of the acid-catalyzed hydrolysis of a hypermodified nucleoside wyosine and its 5'-monophosphate.

    PubMed Central

    Golankiewicz, B; Zielonacka-Lis, E; Folkman, W

    1985-01-01

    The rates of acid-catalyzed hydrolysis of a hypermodified nucleoside, wyosine and its 5'-monophosphate were determined at various pH, temperature and buffer concentrations. The results show that despite distinct differences in structure and the glycosyl bond stability, the hydrolysis of wyosine proceeds via cleavage of the C-N bond by A-1 mechanism, analogously to simple nucleosides. Unlike majority of other monophosphates studied so far, wyosine 5'-monophosphate is not more stable than respective nucleoside. PMID:4000960

  4. Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    PubMed

    Zheng, Zhong-liang; Ye, Mao-qing; Zuo, Zhen-yu; Liu, Zhi-gang; Tai, Keng-chang; Zou, Guo-lin

    2006-05-01

    Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33-->Ala33, Asp60-->Ala60, Ser62-->Ala62, and Thr220-->Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (DeltaDeltaG(T)). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the K(m) values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations. PMID:16411898

  5. Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation

    PubMed Central

    Zheng, Zhong-liang; Ye, Mao-qing; Zuo, Zhen-yu; Liu, Zhi-gang; Tai, Keng-chang; Zou, Guo-lin

    2006-01-01

    Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33→Ala33, Asp60→Ala60, Ser62→Ala62, and Thr220→Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (ΔΔGT). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the Km values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations. PMID:16411898

  6. An RF-Powered Micro-Reactor for Efficient Extraction and Hydrolysis

    NASA Astrophysics Data System (ADS)

    Scott, V.

    2014-12-01

    An RF sample-processing micro-reactor that was developed as part of potential in situ Exploration Missions to inner- and outer-planetary bodies was designed to utilize aqueous solutions subjected to 60 GHz radiation at 730 mW of input power to extract target organic compounds and molecular and inorganic ions as well as to hydrolyze complex polymeric materials. Successful identification and characterization of these molecules relies on the sample-processing techniques utilized alongside state-of-the-art detection and analysis. For mass and power restrictions put on space exploration missions, smaller and more efficient instruments are highly desirable. The RF micro-reactor potentially offers a simplified alternative to the typical gold-standard extractions that often use solvents, chemicals, and conditions that can vary wildly and depend on the targeted molecules. Instead, this instrument uses a single solvent ­— water — that can be "tuned" under the different experimental conditions, leveraging the operating principles of the Sub-Critical Water Extractor. Proof-of-concept experiments examining the hydrolysis of glycosidic and peptide bonds were successful in demonstrating the RF micro-reactor's capabilities. Progress toward coupling the reactor with a micro-scale sample-handling system enabling slurry delivery has been made and preliminary results on heterogeneous reactions and extractions will be presented.

  7. Investigation of the complex reaction coordinate of acid catalyzed amide hydrolysis from molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Zahn, Dirk

    2004-05-01

    The rate-determining step of acid catalyzed peptide hydrolysis is the nucleophilic attack of a water molecule to the carbon atom of the amide group. Therein the addition of the hydroxyl group to the amide carbon atom involves the association of a water molecule transferring one of its protons to an adjacent water molecule. The protonation of the amide nitrogen atom follows as a separate reaction step. Since the nucleophilic attack involves the breaking and formation of several bonds, the underlying reaction coordinate is rather complex. We investigate this reaction step from path sampling Car-Parrinello molecular dynamics simulations. This approach does not require the predefinition of reaction coordinates and is thus particularly suited for investigating reaction mechanisms. From our simulations the most relevant components of the reaction coordinate are elaborated. Though the C⋯O distance of the oxygen atom of the water molecule performing the nucleophilic attack and the corresponding amide carbon atom is a descriptor of the reaction progress, a complete picture of the reaction coordinate must include all three molecules taking part in the reaction. Moreover, the proton transfer is found to depend on favorable solvent configurations. Thus, also the arrangement of non-reacting, i.e. solvent water molecules needs to be considered in the reaction coordinate.

  8. Structure-activity correlations for organophosphorus ester anticholinesterases. Part 2: CNDO/2 calculations applied to ester hydrolysis rates

    NASA Technical Reports Server (NTRS)

    Johnson, H.; Kenley, R. A.; Rynard, C.; Golub, M. A.

    1984-01-01

    Quantitative structure-activity relationships are presented for the hydrolysis of organophosphorus esters, RR'P(O)X, where R and R' are alkyl and/or alkoxy groups and X is fluorine, chlorine or a phenoxy group. CNDO/2 calculations provide values for molecular parameters that correlate with alkaline hydrolysis rates. For each subset of esters with the same leaving group, X, the CNDO-derived net atomic charge at the central phosphorus atom correlates well with the alkaline hydrolysis rate constants. For the whole set of esters with different leaving groups, equations are derived that relate charge, orbital energy and bond order to the hydrolysis rate constants.

  9. Nisin-induced expression of a recombinant antihypertensive peptide in dairy lactic acid bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peptides with antihypertensive activity have been identified from the enzymatic hydrolysis of bovine milk proteins. A 12-residue peptide (FFVAPFPEVFGK) shown to inhibit the angiotensin I-converting enzyme is released from the enzymatic breakdown of aS1-casein. A synthetic gene encoding this peptid...

  10. Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-08-22

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  11. Conformational preferences of heterochiral peptides. Crystal structures of heterochiral peptides Boc-(D) Val-(D) Ala-Leu-Ala-OMe and Boc-Val-Ala-Leu-(D) Ala-OMe--enhanced stability of beta-sheet through C-H...O hydrogen bonds.

    PubMed

    Fabiola, G F; Bobde, V; Damodharan, L; Pattabhi, V; Durani, S

    2001-02-01

    The crystal structures of Boc-(D) Val-(D) Ala-Leu-Ala-OMe (vaLA) and Boc-Val-Ala-Leu-(D) Ala-OMe (VALa) have been determined. vaLA crystallises in space group P2(1),2(1),2(1), with a = 9.401 (4), b = 17.253 (5), c = 36.276 (9)A. V = 5,884 (3) A3, Z = 8, R = 0.086. VALa crystallises in space group P2(1) with a = 9.683 (9), b = 17.355 (7), c = 18.187 (9) A, beta = 95.84 (8) degrees , V = 3,040(4) A3, Z = 4, R = 0.125. There are two molecules in the asymmetric unit in antiparallel beta-sheet arrangement in both the structures. Several of the Calpha hydrogens are in hydrogen bonding contact with the carbonyl oxygen in the adjacent strand. An analysis of the observed conformational feature of D-chiral amino acid residues in oligopeptides, using coordinates of 123 crystal structures selected from the 1998 release of CSD has been carried out. This shows that all the residues except D-isoleucine prefer both extended and alphaL conformation though the frequence of occurence may not be equal. In addition to this, D-leucine, valine, proline and phenylalanine have assumed alphaR conformations in solid state. D-leucine has a strong preference for helical conformation in linear peptides whereas they prefer an extended conformation in cyclic peptides. PMID:11245253

  12. Antimicrobial peptides

    PubMed Central

    2014-01-01

    With increasing antibiotics resistance, there is an urgent need for novel infection therapeutics. Since antimicrobial peptides provide opportunities for this, identification and optimization of such peptides have attracted much interest during recent years. Here, a brief overview of antimicrobial peptides is provided, with focus placed on how selected hydrophobic modifications of antimicrobial peptides can be employed to combat also more demanding pathogens, including multi-resistant strains, without conferring unacceptable toxicity. PMID:24758244

  13. Iron-chelating activity of chickpea protein hydrolysate peptides.

    PubMed

    Torres-Fuentes, Cristina; Alaiz, Manuel; Vioque, Javier

    2012-10-01

    Chickpea-chelating peptides were purified and analysed for their iron-chelating activity. These peptides were purified after affinity and gel filtration chromatography from a chickpea protein hydrolysate produced with pepsin and pancreatin. Iron-chelating activity was higher in purified peptide fractions than in the original hydrolysate. Histidine contents were positively correlated with the iron-chelating activity. Hence fractions with histidine contents above 20% showed the highest chelating activity. These results show that iron-chelating peptides are generated after chickpea protein hydrolysis with pepsin plus pancreatin. These peptides, through metal chelation, may increase iron solubility and bioavailability and improve iron absorption. PMID:25005984

  14. Peptide-formation on cysteine-containing peptide scaffolds

    NASA Technical Reports Server (NTRS)

    Chu, B. C.; Orgel, L. E.

    1999-01-01

    Monomeric cysteine residues attached to cysteine-containing peptides by disulfide bonds can be activated by carbonyldiimidazole. If two monomeric cysteine residues, attached to a 'scaffold' peptide Gly-Cys-Glyn-Cys-Glu10, (n = 0, 1, 2, 3) are activated, they react to form the dipeptide Cys-Cys. in 25-65% yield. Similarly, the activation of a cysteine residue attached to the 'scaffold' peptide Gly-Cys-Gly-Glu10 in the presence of Arg5 leads to the formation of Cys-Arg5 in 50% yield. The significance of these results for prebiotic chemistry is discussed.

  15. HYDROLYSIS OF CHLOROSTILBENE OXIDE: I. HYDROLYSIS IN HOMOGENEOUS SYSTEMS

    EPA Science Inventory

    The hydrolysis kinetics of 4-chlorostilbene oxide (CSO) in buffered distilled water, in natural waters, and in sediment associated water are reported. he disappearance of CSO followed pseudo-first-order kinetics in buffered water over the experimental pH range of 3 to 11. elow pH...

  16. Antiviral active peptide from oyster

    NASA Astrophysics Data System (ADS)

    Zeng, Mingyong; Cui, Wenxuan; Zhao, Yuanhui; Liu, Zunying; Dong, Shiyuan; Guo, Yao

    2008-08-01

    An active peptide against herpes virus was isolated from the enzymic hydrolysate of oyster ( Crassostrea gigas) and purified with the definite direction hydrolysis technique in the order of alcalase and bromelin. The hydrolysate was fractioned into four ranges of molecular weight (>10 kDa, 10 5 kDa, 5 1 kDa and <1 kDa) using ultrafiltration membranes and dialysis. The fraction of 10 5 kDa was purified using consecutive chromatographic methods including DEAE Sephadex A-25 column, Sephadex G-25 column, and high performance liquid chromatogram (HPLC) by activity-guided isolation. The antiviral effect of the obtained peptide on herpetic virus was investigated in Vero cells by observing cytopathic effect (CPE). The result shows that the peptide has high inhibitory activity on herpetic virus.

  17. Evaluation of reduced allergenicity of deamidated gliadin in a mouse model of wheat-gliadin allergy using an antibody prepared by a peptide containing three epitopes.

    PubMed

    Abe, Ryosuke; Shimizu, Shiori; Yasuda, Karin; Sugai, Masae; Okada, Yohei; Chiba, Kazuhiro; Akao, Makoto; Kumagai, Hitoshi; Kumagai, Hitomi

    2014-04-01

    Gliadin is the principal allergen of wheat-dependent exercise-induced anaphylaxis (WDEIA). The primary structure of IgE-binding epitopes in wheat gliadin includes tandem sequencing sites of glutamine residues. Therefore, deamidation would be an effective approach to reduce the allergenicity of wheat proteins. In our previous study, we deamidated wheat gliadin without causing peptide-bond hydrolysis or polymerization by use of carboxylated cation-exchange resins, and we found that the deamidated gliadin scarcely reacted with the sera of patients radioallergosorbent test (RAST)-positive to wheat. In this study, we examined the allergenicity of deamidated gliadin in a mouse model of wheat-gliadin allergy. Oral administration of deamidated gliadin to gliadin-sensitized mice suppressed enhancement in intestinal permeability, serum allergen level, serum allergen-specific IgE level, mast-cell-surface expression of FcεRI, and serum and intestinal histamine levels. Our results indicate that gliadin deamidated with no peptide-bond hydrolysis by cation-exchange resins has low allergenicity even under in vivo conditions. PMID:24617642

  18. Economics of enzymatic hydrolysis processes

    SciTech Connect

    Wright, J.D.

    1988-02-01

    Enzymatic hydrolysis processes have the ability to produce high yields of sugars for fermentation to fuel ethanol from lignocellulosic biomass. However, these systems have been plagued with yields, product concentrations, and reactions rates far below those that are theoretically possible. Engineering and economic analyses are presented on several fungal enzyme hydrolysis processes to illustrate the effects of the important process parameters, to quantify the progress that has been made to date, and to estimate the cost reductions that can be made through research improvements. All enzymatic hydrolysis processes require pretreatment, hydrolysis, fermentation, and enzyme production. The key effect of pretreatment is to allow access of the enzymes to the substrate. Pretreatments have been devised that make the biomass completely digestible that increase the xylose yield and concentration, and that integrate pretreatment with lignin utilization. Major improvements in enzyme activity and use of simultaneous saccharification and fermentation (SSF) have greatly reduced the inhibition of the enzymes. It now appears that ethanol inhibition of the yeast is the limiting factor. Enzyme production costs have been dramatically reduced because use of SSF has reduced enzyme loading. However, further improvements may be possible by using soluble carbon sources for production. Over the past decade, the predicted cost of ethanol from such processes has dropped from more than $4.00/gallon to approximately $1.60. Research is currently under way in the United States and has the potential to reduce the projected cost to less than $1.00/gallon. 65 refs., 16 figs., 1 tab.

  19. Enzymatic hydrolysis of organic phosphorus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Orthophosphate-releasing enzymatic hydrolysis is an alternative means for characterizing organic phosphorus (Po) in animal manure. The approach is not only simple and fast, but can also provide information difficult to obtain by other methods. Currently, commercially available phosphatases are mainl...

  20. Antimicrobial Peptides from Plants.

    PubMed

    Tam, James P; Wang, Shujing; Wong, Ka H; Tan, Wei Liang

    2015-01-01

    Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms. PMID:26580629

  1. Antimicrobial Peptides from Plants

    PubMed Central

    Tam, James P.; Wang, Shujing; Wong, Ka H.; Tan, Wei Liang

    2015-01-01

    Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms. PMID:26580629

  2. Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

    SciTech Connect

    Storrs, R.W.

    1992-08-01

    Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by [sup 31]P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 [Angstrom] of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an [alpha]-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.

  3. Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

    SciTech Connect

    Storrs, R.W.

    1992-08-01

    Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by {sup 31}P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 {Angstrom} of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an {alpha}-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.

  4. Rapid Online Non-Enzymatic Protein Digestion Combining Microwave Heating Acid Hydrolysis and Electrochemical Oxidation

    PubMed Central

    Basile, Franco; Hauser, Nicolas

    2010-01-01

    We report an online non-enzymatic method for site-specific digestion of proteins to yield peptides that are well suited for collision induced dissociation (CID) tandem mass spectrometry (MS/MS). The method combines online microwave heating acid hydrolysis at aspartic acid and online electrochemical oxidation at tryptophan and tyrosine. The combined microwave/electrochemical (microwave/echem) digestion is reproducible and produces peptides with an average sequence length of 10 amino acids. This peptide length is similar to the average peptide length of 9 amino acids obtained by digestion of proteins with the enzyme trypsin. As a result, the peptides produced by this novel non-enzymatic digestion method, when analyzed by ESI-MS, produce protonated molecules with mostly +1 and +2 charge states. The combination of these two non-enzymatic methods overcomes shortcomings with each individual method in that: i) peptides generated by the microwave-hydrolysis method have an average amino acid length of 16 amino acids, and ii) the inability of the electrochemical-cleavage method to reproducibly digest proteins with molecular masses above 4 kDa. Preliminary results are presented on the application and utility of this rapid online digestion (total of 6 min digestion time) on a series of standard peptides and proteins as well as an E. coli protein extract. PMID:21138252

  5. Constrained Peptides as Miniature Protein Structures

    PubMed Central

    Yin, Hang

    2012-01-01

    This paper discusses the recent developments of protein engineering using both covalent and noncovalent bonds to constrain peptides, forcing them into designed protein secondary structures. These constrained peptides subsequently can be used as peptidomimetics for biological functions such as regulations of protein-protein interactions. PMID:25969758

  6. Acid-functionalized nanoparticles for biomass hydrolysis

    NASA Astrophysics Data System (ADS)

    Pena Duque, Leidy Eugenia

    Cellulosic ethanol is a renewable source of energy. Lignocellulosic biomass is a complex material composed mainly of cellulose, hemicellulose, and lignin. Biomass pretreatment is a required step to make sugar polymers liable to hydrolysis. Mineral acids are commonly used for biomass pretreatment. Using acid catalysts that can be recovered and reused could make the process economically more attractive. The overall goal of this dissertation is the development of a recyclable nanocatalyst for the hydrolysis of biomass sugars. Cobalt iron oxide nanoparticles (CoFe2O4) were synthesized to provide a magnetic core that could be separated from reaction using a magnetic field and modified to carry acid functional groups. X-ray diffraction (XRD) confirmed the crystal structure was that of cobalt spinel ferrite. CoFe2O4 were covered with silica which served as linker for the acid functions. Silica-coated nanoparticles were functionalized with three different acid functions: perfluoropropyl-sulfonic acid, carboxylic acid, and propyl-sulfonic acid. Transmission electron microscope (TEM) images were analyzed to obtain particle size distributions of the nanoparticles. Total carbon, nitrogen, and sulfur were quantified using an elemental analyzer. Fourier transform infra-red spectra confirmed the presence of sulfonic and carboxylic acid functions and ion-exchange titrations accounted for the total amount of catalytic acid sites per nanoparticle mass. These nanoparticles were evaluated for their performance to hydrolyze the beta-1,4 glycosidic bond of the cellobiose molecule. Propyl-sulfonic (PS) and perfluoropropyl-sulfonic (PFS) acid functionalized nanoparticles catalyzed the hydrolysis of cellobiose significantly better than the control. PS and PFS were also evaluated for their capacity to solubilize wheat straw hemicelluloses and performed better than the control. Although PFS nanoparticles were stronger acid catalysts, the acid functions leached out of the nanoparticle during

  7. Bond Issues.

    ERIC Educational Resources Information Center

    Pollack, Rachel H.

    2000-01-01

    Notes trends toward increased borrowing by colleges and universities and offers guidelines for institutions that are considering issuing bonds to raise money for capital projects. Discussion covers advantages of using bond financing, how use of bonds impacts on traditional fund raising, other cautions and concerns, and some troubling aspects of…

  8. Reductive cleavage of the peptide bond

    NASA Technical Reports Server (NTRS)

    Holian, J.; Garrison, W. M.

    1973-01-01

    In many biological research efforts, long chain organic molecules are studied by breaking large molecules into smaller components. Cleavage technique of recent interest is the use of solvated electrons. These are formed when aqueous solutions are bombarded with gamma radiation. Solvated electron is very reactive and can reduce most any species present, even to form free radicals.

  9. Kinetics and thermodynamics of sucrose hydrolysis from real-time enthalpy and heat capacity measurements.

    PubMed

    Tombari, E; Salvetti, G; Ferrari, C; Johari, G P

    2007-01-25

    We report a real time study of the enthalpy release and heat capacity during the course of HCl-catalyzed hydrolysis of sucrose to fructose and glucose. Measurements were performed during both isothermal conditions and during slow heating and then cooling at a controlled rate. The reaction rate constant of the first-order kinetics follows an Arrhenius relation with activation energy of 109.2 kJ/mol of sucrose. On hydrolysis, the enthalpy decreases by 14.4 kJ/mol of sucrose at 310 K, and the heat capacity, Cp, increases by 61 J mol-1 K-1 of sucrose in the solution. The enthalpy of hydrolysis decreases with increase in the temperature and DeltaCp on hydrolysis increases. The effects are attributed to change in the configurational and vibrational partition functions as one covalent bond in sucrose breaks to form two molecules, which then individually form additional hydrogen bonds and alter the water's structure in the solution. Cp of the solution increases with temperature less rapidly before sucrose hydrolysis than after it. This may reflect an increase in the configurational contribution to Cp as the hydrogen bond population changes. PMID:17228904

  10. Generation of group B soyasaponins I and III by hydrolysis.

    PubMed

    Zhang, Wei; Teng, Su Ping; Popovich, David G

    2009-05-13

    Soyasaponins are a group of oleanane triterpenoids found in soy and other legumes that have been associated with some of the benefits achieved by consuming plant-based diets. However, these groups of compounds are diverse and structurally complicated to chemically characterize, separate from the isoflavones, and isolate in sufficient quantities for bioactive testing. Therefore, the aim of this study was to maximize the extraction of soyasaponins from soy flour, remove isoflavones, separate group B soyasaponins from group A, and produce an extract that contained a majority of non-DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one)-conjugated group B soyasaponins I and III. Room temperature extraction in methanol for 24 or 48 h resulted in the maximum recovery of soyasaponins, and Soxhlet extraction resulted in the least. A solid-phase extraction using methanol (45%) was found to virtually eliminate the interfering isoflavones as compared to butanol-water liquid-liquid extraction and ammonium sulfate precipitation, while maximizing saponin recovery. Alkaline hydrolysis in anhydrous methanol produced the maximum amount of soyasaponins I and III as compared to aqueous methanol and acid hydrolysis in both aqueous and anhydrous methanol. The soyasaponin I amount was increased by 175%, and soyasaponin III was increased by 211% after alkaline hydrolysis. Furthermore, after alkaline hydrolysis, a majority of DDMP-conjugated group B soyasaponins such as betag, betaa, gammag, and gammaa transformed into the non-DDMP-conjugated soyasaponins I and III without affecting the glycosidic bond at position C-3 of the ring structure. Therefore, we have developed a method that maximizes the recovery of DDMP-conjugated saponins and uses alkaline hydrolysis to produce an extract containing mainly soyasaponins I and III. PMID:19338335

  11. Structure and hydrolysis of p-(2-oxo-1-pyrrolidinyl)- benzenesulfonic acid

    SciTech Connect

    Kukalenko, S.S.; Frolov, S.I.; Lim, I.K.; Putsykina, E.B.; Vasil'ev, A.F.

    1987-11-20

    With the aid of vibrational and PMR spectra of p-(2-oso-1-pyrrolidinyl)benzenesulfonic acid it was shown that in the solid state it exists as an O-protonated dipolar ion in which the protonated amide cation and sulfonate ion are intermolecularly linked by a very strong hydrogen bond. In concentrated hydrochloric acid the dipolar ion is an intermediate link in the chain of processes in the hydrolysis of the amide bond of the lactam ring.

  12. Fatty acid hydrolysis of acyl marinobactin siderophores by Marinobacter acylases.

    PubMed

    Kem, Michelle P; Naka, Hiroaki; Iinishi, Akira; Haygood, Margo G; Butler, Alison

    2015-01-27

    The marine bacteria Marinobacter sp. DS40M6 and Marinobacter nanhaiticus D15-8W produce a suite of acyl peptidic marinobactin siderophores to acquire iron under iron-limiting conditions. During late-log phase growth, the marinobactins are hydrolyzed to form the marinobactin headgroup with release of the corresponding fatty acid tail. The bntA gene, a homologue of the Pseudomonas aeruginosa pyoverdine acylase gene, pvdQ, was identified from Marinobacter sp. DS40M6. A bntA knockout mutant of Marinobacter sp. DS40M6 produced the suite of acyl marinobactins A-E, without the usual formation of the marinobactin headgroup. Another marinobactin-producing species, M. nanhaiticus D15-8W, is predicted to have two pvdQ homologues, mhtA and mhtB. MhtA and MhtB have 67% identical amino acid sequences. MhtA catalyzes hydrolysis of the apo-marinobactin siderophores as well as the quorum sensing signaling molecule, dodecanoyl-homoserine lactone. In contrast to hydrolysis of the suite of apo-marinobactins by MhtA, hydrolysis of the iron(III)-bound marinobactins was not observed. PMID:25588131

  13. Lignin recovery from alkaline hydrolysis and glycerolysis of oil palm fiber

    NASA Astrophysics Data System (ADS)

    Hassan, Nur Syakilla; Badri, Khairiah Haji

    2014-09-01

    In the present work, two types of treatment namely alkaline hydrolysis and glycerolysis have been conducted for lignin extraction from oil palm empty fruit bunch (EFB) fiber. Lignin has been retrieved from two sequential methods, which was the klason lignin from residue and lignin from precipitation of the filtrate. Alkaline hydrolysis was performed using 10% NaOH solution at room condition. This has extracted 13.0 % lignin. On the other hand, glycerolysis was carried out using 70% glycerol catalyzed with 5% of 1 M NaOH at 60-70 °C. This has successfully extracted 16.0% lignin. The SEM micrographs exhibited some physical changes on the surface where the impurities and waxes have been removed, exposing the, lumen. Besides that, FTIR analysis was conducted on untreated EFB, treated EFB and extracted lignin. Delignification of EFB fiber was confirmed based on the intensity reduction at 1245 cm-1 that showed lignin was removed from the fiber. The presence of CO, CC and CC aromatic peaks in the FTIR spectra of the dried filtrate gave an evidence on the presence of lignin.

  14. Comparison of Enzymatic Hydrolysis and Acid Hydrolysis of Sterol Glycosides from Foods Rich in Δ(7)-Sterols.

    PubMed

    Münger, Linda H; Jutzi, Sabrina; Lampi, Anna-Maija; Nyström, Laura

    2015-08-01

    In this study, we present the difference in sterol composition of extracted steryl glycosides (SG) hydrolyzed by either enzymatic or acid hydrolysis. SG were analyzed from foods belonging to the plant families Cucurbitaceae (melon and pumpkin seeds) and Amaranthaceae (amaranth and beetroot), both of which are dominated by Δ(7)-sterols. Released sterols were quantified by gas chromatography with a flame ionization detector (GC-FID) and identified using gas chromatography/mass spectrometry (GC-MS). All Δ(7)-sterols identified (Δ(7)-stigmastenyl, spinasteryl, Δ(7)-campesteryl, Δ(7)-avenasteryl, poriferasta-7,25-dienyl and poriferasta-7,22,25-trienyl glucoside) underwent isomerization under acidic conditions and high temperature. Sterols with an ethylidene or methylidene side chain were found to form multiple artifacts. The artifact sterols coeluted with residues of incompletely isomerized Δ(7)-sterols, or Δ(5)-sterols if present, and could be identified as Δ(8(14))-sterols on the basis of relative retention time, and their MS spectra as trimethylsilyl (TMS) and acetate derivatives. For instance, SG from melon were composed of 66% Δ(7)-stigmastenol when enzymatic hydrolysis was performed, whereas with acid hydrolysis only 8% of Δ(7)-stigmastenol was determined. The artifact of Δ(7)-stigmastenol coeluted with residual non-isomerized spinasterol, demonstrating the high risk of misinterpretation of compositional data obtained after acid hydrolysis. Therefore, the accurate composition of SG from foods containing sterols with a double bond at C-7 can only be obtained by enzymatic hydrolysis or by direct analysis of the intact SG. PMID:25757602

  15. High Resolution Structures of p-Aminobenzamidine- and Benzamidine- VIIa/Soluble Tissue Factor: Unpredicted Conformation of the 192-193 Peptide Bond and Mapping of Ca2+, Mg2+, Na+ an Zn2+ Sites in Facto VIIa

    SciTech Connect

    Bajaj,S.; Schmidt, A.; Agah, S.; Bajaj, M.; Padmanabhan, K.

    2006-01-01

    Factor VIIa (FVIIa) consists of a {gamma}-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca{sup 2+} ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca{sup 2+} and Mg{sup 2+}, and the Ca{sup 2+} sites in FVIIa that could be specifically occupied by Mg{sup 2+} are unknown. Furthermore, FVIIa contains a Na{sup +} and two Zn{sup 2+} sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca{sup 2+}, Mg{sup 2+}, Na{sup +}, and Zn{sup 2+}. The crystal diffracted to 1.8{angstrom} resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca{sup 2+} and three bound Mg{sup 2+}. The EGF1 domain contains one Ca{sup 2+} site, and the protease domain contains one Ca{sup 2+}, one Na{sup +}, and two Zn{sup 2+} sites. {sup 45}Ca{sup 2+} binding in the presence/absence of Mg{sup 2+} to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly{sup 193} in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87{angstrom} resolution. However, soaking benzamidine-FVIIa/sTF crystals with D-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, D-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.

  16. Characterization of Peptides Found in Unprocessed and Extruded Amaranth (Amaranthus hypochondriacus) Pepsin/Pancreatin Hydrolysates

    PubMed Central

    Montoya-Rodríguez, Alvaro; Milán-Carrillo, Jorge; Reyes-Moreno, Cuauhtémoc; González de Mejía, Elvira

    2015-01-01

    The objectives of this study were to characterize peptides found in unprocessed amaranth hydrolysates (UAH) and extruded amaranth hydrolysates (EAH) and to determine the effect of the hydrolysis time on the profile of peptides produced. Amaranth grain was extruded in a single screw extruder at 125 °C of extrusion temperature and 130 rpm of screw speed. Unprocessed and extruded amaranth flour were hydrolyzed with pepsin/pancreatin enzymes following a kinetic at 10, 25, 60, 90, 120 and 180 min for each enzyme. After 180 min of pepsin hydrolysis, aliquots were taken at each time during pancreatin hydrolysis to characterize the hydrolysates by MALDI-TOF/MS-MS. Molecular masses (MM) (527, 567, 802, 984, 1295, 1545, 2034 and 2064 Da) of peptides appeared consistently during hydrolysis, showing high intensity at 10 min (2064 Da), 120 min (802 Da) and 180 min (567 Da) in UAH. EAH showed high intensity at 10 min (2034 Da) and 120 min (984, 1295 and 1545 Da). Extrusion produced more peptides with MM lower than 1000 Da immediately after 10 min of hydrolysis. Hydrolysis time impacted on the peptide profile, as longer the time lower the MM in both amaranth hydrolysates. Sequences obtained were analyzed for their biological activity at BIOPEP, showing important inhibitory activities related to chronic diseases. These peptides could be used as a food ingredient/supplement in a healthy diet to prevent the risk to develop chronic diseases. PMID:25894223

  17. Characterization of peptides found in unprocessed and extruded amaranth (Amaranthus hypochondriacus) pepsin/pancreatin hydrolysates.

    PubMed

    Montoya-Rodríguez, Alvaro; Milán-Carrillo, Jorge; Reyes-Moreno, Cuauhtémoc; González de Mejía, Elvira

    2015-01-01

    The objectives of this study were to characterize peptides found in unprocessed amaranth hydrolysates (UAH) and extruded amaranth hydrolysates (EAH) and to determine the effect of the hydrolysis time on the profile of peptides produced. Amaranth grain was extruded in a single screw extruder at 125 °C of extrusion temperature and 130 rpm of screw speed. Unprocessed and extruded amaranth flour were hydrolyzed with pepsin/pancreatin enzymes following a kinetic at 10, 25, 60, 90, 120 and 180 min for each enzyme. After 180 min of pepsin hydrolysis, aliquots were taken at each time during pancreatin hydrolysis to characterize the hydrolysates by MALDI-TOF/MS-MS. Molecular masses (MM) (527, 567, 802, 984, 1295, 1545, 2034 and 2064 Da) of peptides appeared consistently during hydrolysis, showing high intensity at 10 min (2064 Da), 120 min (802 Da) and 180 min (567 Da) in UAH. EAH showed high intensity at 10 min (2034 Da) and 120 min (984, 1295 and 1545 Da). Extrusion produced more peptides with MM lower than 1000 Da immediately after 10 min of hydrolysis. Hydrolysis time impacted on the peptide profile, as longer the time lower the MM in both amaranth hydrolysates. Sequences obtained were analyzed for their biological activity at BIOPEP, showing important inhibitory activities related to chronic diseases. These peptides could be used as a food ingredient/supplement in a healthy diet to prevent the risk to develop chronic diseases. PMID:25894223

  18. HYDROLYSIS

    EPA Science Inventory

    Hydrolytic processes provide the baseline loss rate for any chemical in an aqueous envi- ronment. Although various hydrolytic pathways account for significant degradation of certain classes of organic chemicals, other organic structures are completely inert. Strictly speaking, hy...

  19. Identification of short peptide sequences in complex milk protein hydrolysates.

    PubMed

    O'Keeffe, Martina B; FitzGerald, Richard J

    2015-10-01

    Numerous low molecular mass bioactive peptides (BAPs) can be generated during the hydrolysis of bovine milk proteins. Low molecular mass BAP sequences are less likely to be broken down by digestive enzymes and are thus more likely to be active in vivo. However, the identification of short peptides remains a challenge during mass spectrometry (MS) analysis due to issues with the transfer and over-fragmentation of low molecular mass ions. A method is described herein using time-of-flight ESI-MS/MS to effectively fragment and identify short peptides. This includes (a) short synthetic peptides, (b) short peptides within a defined hydrolysate sample, i.e. a prolyl endoproteinase hydrolysate of β-casein and (c) short peptides within a complex hydrolysate, i.e. a Corolase PP digest of sodium caseinate. The methodology may find widespread utilisation in the efficient identification of low molecular mass peptide sequences in food protein hydrolysates. PMID:25872436

  20. Superior Antifouling Performance of a Zwitterionic Peptide Compared to an Amphiphilic, Non-Ionic Peptide.

    PubMed

    Ye, Huijun; Wang, Libing; Huang, Renliang; Su, Rongxin; Liu, Boshi; Qi, Wei; He, Zhimin

    2015-10-14

    The aim of this study was to explore the influence of amphiphilic and zwitterionic structures on the resistance of protein adsorption to peptide self-assembled monolayers (SAMs) and gain insight into the associated antifouling mechanism. Two kinds of cysteine-terminated heptapeptides were studied. One peptide had alternating hydrophobic and hydrophilic residues with an amphiphilic sequence of CYSYSYS. The other peptide (CRERERE) was zwitterionic. Both peptides were covalently attached onto gold substrates via gold-thiol bond formation. Surface plasmon resonance analysis results showed that both peptide SAMs had ultralow or low protein adsorption amounts of 1.97-11.78 ng/cm2 in the presence of single proteins. The zwitterionic peptide showed relatively higher antifouling ability with single proteins and natural complex protein media. We performed molecular dynamics simulations to understand their respective antifouling behaviors. The results indicated that strong surface hydration of peptide SAMs contributes to fouling resistance by impeding interactions with proteins. Compared to the CYSYSYS peptide, more water molecules were predicted to form hydrogen-bonding interactions with the zwitterionic CRERERE peptide, which is in agreement with the antifouling test results. These findings reveal a clear relation between peptide structures and resistance to protein adsorption, facilitating the development of novel peptide-containing antifouling materials. PMID:26407144

  1. Effect of exopolysaccharides on the hydrolysis of beta-lactoglobulin by Lactobacillus acidophilus CRL 636 in an in vitro gastric/pancreatic system.

    PubMed

    Pescuma, Micaela; Hébert, Elvira María; Dalgalarrondo, Michèle; Haertlé, Thomas; Mozzi, Fernanda; Chobert, Jean-Marc; Font de Valdez, Graciela

    2009-06-24

    An analysis of the peptides generated by hydrolysis of BLG by nonproliferating cells of the strain Lactobacillus acidophilus CRL 636 was carried out. The effect of polysaccharides (pectin, and two EPS synthesized by two Streptococcus thermophilus strains, EPS1190 and EPS804) on BLG digestibility using an in vitro gastric/pancreatic system was analyzed. Polysaccharides are commonly used in the dairy industry to improve food texture; these hydrocolloids may interact with proteins, affecting their digestibility. Nonproliferating cells of Lb. acidophilus CRL 636 were able to hydrolyze 52% of BLG. Twenty-six resulting peptides with molecular masses in the range 544-4119 Da were identified by LC-MS/MS. These peptides resulted mostly from the hydrolysis of the more accessible N-terminal part of BLG. Degradation of BLG by pepsin was poor (8%). When BLG was previously hydrolyzed by Lb. acidophilus CRL 636, peptic hydrolysis was of 54.8%, while when pectin and EPS1190 were added, hydrolysis was higher (58.2 and 57.2%, respectively). Peptides crossing 8 kDa dialysis membranes after trypsin/chymotrypsin hydrolysis were analyzed by HPSEC. The produced peptides were smaller when BLG was hydrolyzed previously by the Lb. acidophilus strain. Moreover, in the presence of pectin, the amount of the larger peptide (3.5 kDa) observed in the size exclusion chromatograms was considerably decreased. Our studies showed that prehydrolysis of BLG by Lb. acidophilus CRL 636 had a positive influence on BLG digestibility and that polysaccharides may change the peptide profile yielded by trypsin/chymotrypsin hydrolysis, releasing smaller size peptides, which are known to be less immune-reactive. Moreover, Lb. acidophilus CRL 636 was able to hydrolyze the main epitopes (41-60, 102-124, and 149-162) of BLG, reducing its allergenic content. PMID:19469473

  2. Protein inhibitors of serine proteinases: role of backbone structure and dynamics in controlling the hydrolysis constant.

    PubMed

    Song, Jikui; Markley, John L

    2003-05-13

    Standard mechanism protein inhibitors of serine proteinases bind as substrates and are cleaved by cognate proteinases at their reactive sites. The hydrolysis constant for this cleavage reaction at the P(1)-P(1)' peptide bond (K(hyd)) is determined by the relative concentrations at equilibrium of the "intact" (uncleaved, I) and "modified" (reactive site cleaved, I*) forms of the inhibitor. The pH dependence of K(hyd) can be explained in terms of a pH-independent term, K(hyd) degrees, plus the proton dissociation constants of the newly formed amino and carboxylate groups at the cleavage site. Two protein inhibitors that differ from one another by a single residue substitution have been found to have K(hyd) degrees values that differ by a factor of 5 [Ardelt, W., and Laskowski, M., Jr. (1991) J. Mol. Biol. 220, 1041-1052]: turkey ovomucoid third domain (OMTKY3) has K(hyd) degrees = 1.0, and Indian peafowl ovomucoid third domain (OMIPF3), which differs from OMTKY3 by the substitution P(2)'-Tyr(20)His, has K(hyd) degrees = 5.15. What mechanism is responsible for this small difference? Is it structural (enthalpic) or dynamic (entropic)? Does the mutation affect the free energy of the I state, the I* state, or both? We have addressed these questions through NMR investigations of the I and I forms of OMTKY3 and OMIPF3. Information about structure was derived from measurements of NMR chemical shift changes and trans-hydrogen-bond J-couplings; information about dynamics was obtained through measurements of (15)N relaxation rates and (1)H-(15)N heteronuclear NOEs with model-free analysis of the results. Although the I forms of each variant are more dynamic than the corresponding I forms, the study revealed no appreciable difference in the backbone dynamics of either intact inhibitor (OMIPF3 vs OMTKY3) or modified inhibitor (OMIPF3* vs OMTKY3*). Instead, changes in chemical shifts and trans-hydrogen-bond J-couplings suggested that the K(hyd) degrees difference arises from

  3. The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis

    PubMed Central

    2016-01-01

    Despite the numerous experimental and theoretical studies on phosphate monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for phosphate monoester hydrolysis to explore the effect of modulating leaving group pKa on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl phosphate and a series of substituted aryl phosphate monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pKa increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations show that the transition-state structures vary smoothly across the range of pKas studied and that the pathways remain discrete mechanistic alternatives. Therefore, while not impossible, a biological catalyst would have to surmount a significantly higher activation barrier to facilitate a substrate-assisted pathway than for the solvent-assisted pathway when phosphate is bonded to good leaving groups. For poor leaving groups, this intrinsic preference disappears. PMID:27471914

  4. Institutional Bonding.

    ERIC Educational Resources Information Center

    Allard, M. June

    Institutional bonding was examined at a public, urban commuter college with exceptionally high attrition and visibly low morale. Changes in bonding and attrition were measured 6 years after a 2-year effort to develop school identity and student feelings of membership. It was found that a simple index of campus morale is provided by level of…

  5. New antibacterial peptide derived from bovine hemoglobin.

    PubMed

    Daoud, Rachid; Dubois, Veronique; Bors-Dodita, Loredana; Nedjar-Arroume, Naima; Krier, Francois; Chihib, Nour-Eddine; Mary, Patrice; Kouach, Mostafa; Briand, Gilbert; Guillochon, Didier

    2005-05-01

    Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration. PMID:15808900

  6. Peptide identification

    DOEpatents

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  7. Acid hydrolysis of cellulose to yield glucose

    DOEpatents

    Tsao, George T.; Ladisch, Michael R.; Bose, Arindam

    1979-01-01

    A process to yield glucose from cellulose through acid hydrolysis. Cellulose is recovered from cellulosic materials, preferably by pretreating the cellulosic materials by dissolving the cellulosic materials in Cadoxen or a chelating metal caustic swelling solvent and then precipitating the cellulose therefrom. Hydrolysis is accomplished using an acid, preferably dilute sulfuric acid, and the glucose is yielded substantially without side products. Lignin may be removed either before or after hydrolysis.

  8. Switching catalysis from hydrolysis to perhydrolysis in Pseudomonas fluorescens esterase.

    PubMed

    Yin, De Lu Tyler; Bernhardt, Peter; Morley, Krista L; Jiang, Yun; Cheeseman, Jeremy D; Purpero, Vincent; Schrag, Joseph D; Kazlauskas, Romas J

    2010-03-01

    Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of epsilon-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher k(cat), but K(m) also increased so the specificity constant, k(cat)/K(m), remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of epsilon-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access

  9. Switching Catalysis from Hydrolysis to Perhydrolysis in Pseudomonas fluorescens Esterase

    SciTech Connect

    Yin, D.; Bernhardt, P; Morley, K; Jiang, Y; Cheeseman, J; Purpero, V; Schrag, J; Kazlauskas, R

    2010-01-01

    Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of {var_epsilon}-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher k{sub cat}, but K{sub m} also increased so the specificity constant, k{sub cat}/K{sub m}, remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of {var_epsilon}-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the

  10. Acid Hydrolysis of Trioxalatocobaltate (III) Ion

    ERIC Educational Resources Information Center

    Wiggans, P. W.

    1975-01-01

    Describes an investigation involving acid hydrolysis and using both volumetric and kinetic techniques. Presents examples of the determination of the rate constant and its variation with temperature. (GS)

  11. Contrasting effects of hardwood and softwood organosolv lignins on enzymatic hydrolysis of lignocellulose.

    PubMed

    Lai, Chenhuan; Tu, Maobing; Shi, Zhiqiang; Zheng, Ke; Olmos, Luis G; Yu, Shiyuan

    2014-07-01

    Identifying an appropriate parameter to elucidate effects of lignin on enzymatic hydrolysis is essential to understand the interactions between enzymes and lignin. Contrasting effects of hardwood organosolv lignin (EOL-SG) and softwood organosolv lignin (EOL-LP) on enzymatic hydrolysis were observed. The addition of EOL-SG (8 g/L) significantly improved the 72 h hydrolysis yields of organosolv pretreated sweetgum (OPSG) and loblolly pine (OPLP) from 49.3% to 68.6% and from 41.2% to 60.8%, respectively. In contrast, the addition of EOL-LP decreased the 72 h hydrolysis yields of OPSG and OPLP to 42.0% and 38.1%, respectively. A strong correlation between the distribution coefficients of cellulase enzymes on lignins and the changes of hydrolysis yields indicated that the inhibitory or stimulatory effects of organosolv lignins on enzymatic hydrolysis were governed by the distribution coefficients (R). The different R values probably were related to the electrostatic interactions, hydrophobic interactions and hydrogen bondings between enzymes and lignin. PMID:24835744

  12. Major peptides from amaranth (Amaranthus cruentus) protein inhibit HMG-CoA reductase activity.

    PubMed

    Soares, Rosana Aparecida Manólio; Mendonça, Simone; de Castro, Luíla Ívini Andrade; Menezes, Amanda Caroline Cardoso Corrêa Carlos; Arêas, José Alfredo Gomes

    2015-01-01

    The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect. PMID:25690031

  13. Major Peptides from Amaranth (Amaranthus cruentus) Protein Inhibit HMG-CoA Reductase Activity

    PubMed Central

    Soares, Rosana Aparecida Manólio; Mendonça, Simone; de Castro, Luíla Ívini Andrade; Menezes, Amanda Caroline Cardoso Corrêa Carlos; Arêas, José Alfredo Gomes

    2015-01-01

    The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect. PMID:25690031

  14. Rate of Hydrolysis of Tertiary Halogeno Alkanes

    ERIC Educational Resources Information Center

    Pritchard, D. R.

    1978-01-01

    Describes an experiment to measure the relative rate of hydrolysis of the 2-x-2 methylpropanes, where x is bromo, chloro or iodo. The results are plotted on a graph from which the relative rate of hydrolysis can be deduced. (Author/GA)

  15. Microwave Pretreatment For Hydrolysis Of Cellulose

    NASA Technical Reports Server (NTRS)

    Cullingford, Hatice S.; George, Clifford E.; Lightsey, George R.

    1993-01-01

    Microwave pretreatment enhances enzymatic hydrolysis of cellulosic wastes into soluble saccharides used as feedstocks for foods, fuels, and other products. Low consumption of energy, high yield, and low risk of proposed hydrolysis process incorporating microwave pretreatment makes process viable alternative to composting.

  16. Latarcins: versatile spider venom peptides.

    PubMed

    Dubovskii, Peter V; Vassilevski, Alexander A; Kozlov, Sergey A; Feofanov, Alexey V; Grishin, Eugene V; Efremov, Roman G

    2015-12-01

    Arthropod venoms feature the presence of cytolytic peptides believed to act synergetically with neurotoxins to paralyze prey or deter aggressors. Many of them are linear, i.e., lack disulfide bonds. When isolated from the venom, or obtained by other means, these peptides exhibit common properties. They are cationic; being mostly disordered in aqueous solution, assume amphiphilic α-helical structure in contact with lipid membranes; and exhibit general cytotoxicity, including antifungal, antimicrobial, hemolytic, and anticancer activities. To suit the pharmacological needs, the activity spectrum of these peptides should be modified by rational engineering. As an example, we provide a detailed review on latarcins (Ltc), linear cytolytic peptides from Lachesana tarabaevi spider venom. Diverse experimental and computational techniques were used to investigate the spatial structure of Ltc in membrane-mimicking environments and their effects on model lipid bilayers. The antibacterial activity of Ltc was studied against a panel of Gram-negative and Gram-positive bacteria. In addition, the action of Ltc on erythrocytes and cancer cells was investigated in detail with confocal laser scanning microscopy. In the present review, we give a critical account of the progress in the research of Ltc. We explore the relationship between Ltc structure and their biological activity and derive molecular characteristics, which can be used for optimization of other linear peptides. Current applications of Ltc and prospective use of similar membrane-active peptides are outlined. PMID:26286896

  17. Construction of Lasso Peptide Fusion Proteins.

    PubMed

    Zong, Chuhan; Maksimov, Mikhail O; Link, A James

    2016-01-15

    Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) typified by an isopeptide-bonded macrocycle between the peptide N-terminus and an aspartate or glutamate side chain. The C-terminal portion of the peptide threads through the N-terminal macrocycle to give the characteristic lasso fold. Because of the inherent stability, both proteolytic and often thermal, of lasso peptides, we became interested in whether proteins could be fused to the free C-terminus of lasso peptides. Here, we demonstrate fusion of two model proteins, the artificial leucine zipper A1 and the superfolder variant of GFP, to the C-terminus of the lasso peptide astexin-1. Successful lasso cyclization of the N-terminus of these fusion proteins requires a flexible linker in between the C-terminus of the lasso peptide and the N-terminus of the protein of interest. The ability to fuse lasso peptides to a protein of interest is an important step toward phage and bacterial display systems for the high-throughput screening of lasso peptide libraries for new functions. PMID:26492187

  18. Effects of partial hydrolysis and subsequent cross-linking on wheat gluten physicochemical properties and structure.

    PubMed

    Wang, Kaiqiang; Luo, Shuizhong; Cai, Jing; Sun, Qiaoqiao; Zhao, Yanyan; Zhong, Xiyang; Jiang, Shaotong; Zheng, Zhi

    2016-04-15

    The rheological behavior and thermal properties of wheat gluten following partial hydrolysis using Alcalase and subsequent microbial transglutaminase (MTGase) cross-linking were investigated. The wheat gluten storage modulus (G') and thermal denaturation temperature (Tg) were significantly increased from 2.26 kPa and 54.43°C to 7.76 kPa and 57.69°C, respectively, by the combined action of partial hydrolysis (DH 0.187%) and cross-linking. The free SH content, surface hydrophobicity, and secondary structure analysis suggested that an appropriate degree of Alcalase-based hydrolysis allowed the compact wheat gluten structure to unfold, increasing the β-sheet content and surface hydrophobicity. This improved its molecular flexibility and exposed additional glutamine sites for MTGase cross-linking. SEM images showed that a compact 3D network formed, while SDS-PAGE profiles revealed that excessive hydrolysis resulted in high-molecular-weight subunits degrading to smaller peptides, unsuitable for cross-linking. It was also demonstrated that the combination of Alcalase-based partial hydrolysis with MTGase cross-linking might be an effective method for modifying wheat gluten rheological behavior and thermal properties. PMID:26616937

  19. Electron Transport in Short Peptide Single Molecules

    NASA Astrophysics Data System (ADS)

    Cui, Jing; Brisendine, Joseph; Ng, Fay; Nuckolls, Colin; Koder, Ronald; Venkarataman, Latha

    We present a study of the electron transport through a series of short peptides using scanning tunneling microscope-based break junction method. Our work is motivated by the need to gain a better understanding of how various levels of protein structure contribute to the remarkable capacity of proteins to transport charge in biophysical processes such as respiration and photosynthesis. We focus here on short mono, di and tri-peptides, and probe their conductance when bound to gold electrodes in a native buffer environment. We first show that these peptides can bind to gold through amine, carboxyl, thiol and methyl-sulfide termini. We then focus on two systems (glycine and alanine) and show that their conductance decays faster than alkanes terminated by the same linkers. Importantly, our results show that the peptide bond is less conductive than a sigma carbon-carbon bond. This work was supported in part by NSF-DMR 1507440.

  20. Antioxidant peptides from corn gluten meal: Orthogonal design evaluation.

    PubMed

    Zhou, Cunshan; Hu, Jiali; Ma, Haile; Yagoub, Abu ElGasim A; Yu, Xiaojie; Owusu, John; Ma, Haiyan; Qin, Xiaopei

    2015-11-15

    Protamex catalyzed corn gluten meal (CGM) hydrolysis peptides (CHP) were prepared. Orthogonal design L16 (4(5)) was used to optimize processing variables of CGM concentration, CGM heat pretreatment (121 °C) time, and enzymolysis pH, temperature, and time. Degree of hydrolysis (DH), undigested residue ratio, molecular weight (MW) distribution and DPPH radical inhibition were selected as analysis indicators. Optimum variables were CGM concentration of 18%, heat pretreatment time of 40 min, and enzymolysis pH, temperature and time of 7.5, 55 °C and 24h, respectively. Verification test showed that CHP IC50 for scavenging hydroxyl radical was the best and then followed by reducing power. Oligopeptides improved after hydrolysis at the expense of di- and tripeptides, suggesting formation of soluble aggregates from low MW peptides. The increase in the DH, oligopeptides, Alanyl-Tyrosine, and antioxidant free amino acids coincided with the improvement in the antioxidant activity of CHP. PMID:25977026

  1. Distinguishing Bonds.

    PubMed

    Rahm, Martin; Hoffmann, Roald

    2016-03-23

    The energy change per electron in a chemical or physical transformation, ΔE/n, may be expressed as Δχ̅ + Δ(VNN + ω)/n, where Δχ̅ is the average electron binding energy, a generalized electronegativity, ΔVNN is the change in nuclear repulsions, and Δω is the change in multielectron interactions in the process considered. The last term can be obtained by the difference from experimental or theoretical estimates of the first terms. Previously obtained consequences of this energy partitioning are extended here to a different analysis of bonding in a great variety of diatomics, including more or less polar ones. Arguments are presented for associating the average change in electron binding energy with covalence, and the change in multielectron interactions with electron transfer, either to, out, or within a molecule. A new descriptor Q, essentially the scaled difference between the Δχ̅ and Δ(VNN + ω)/n terms, when plotted versus the bond energy, separates nicely a wide variety of bonding types, covalent, covalent but more correlated, polar and increasingly ionic, metallogenic, electrostatic, charge-shift bonds, and dispersion interactions. Also, Q itself shows a set of interesting relations with the correlation energy of a bond. PMID:26910496

  2. Marine algae-derived bioactive peptides for human nutrition and health.

    PubMed

    Fan, Xiaodan; Bai, Lu; Zhu, Liang; Yang, Li; Zhang, Xuewu

    2014-09-24

    Within the parent protein molecule, most peptides are inactive, and they are released with biofunctionalities after enzymatic hydrolysis. Marine algae have high protein content, up to 47% of the dry weight, depending on the season and the species. Recently, there is an increasing interest in using marine algae protein as a source of bioactive peptides due to their health promotion and disease therapy potentials. This review presents an overview of marine algae-derived bioactive peptides and especially highlights some key issues, such as in silico proteolysis and quantitative structure-activity relationship studies, in vivo fate of bioactive peptides, and novel technologies in bioactive peptides studies and production. PMID:25179496

  3. Bond distances in polypeptide backbones depend on the local conformation.

    PubMed

    Improta, Roberto; Vitagliano, Luigi; Esposito, Luciana

    2015-06-01

    By combining quantum-mechanical analysis of small model peptides and statistical surveys of high-resolution protein structures, a systematic conformational dependence of bond lengths in polypeptide backbones has been unveiled which involves both the peptide bond (C-O and C-N) and those bonds centred on the C(α) atom. All of these bond lengths indeed display a systematic variability in the ψ angle according to both calculations and surveys of protein structures. The overall agreement between the computed and the statistical data suggests that these trends are essentially driven by local effects. The dependence of C(α) distances on ψ is governed by interactions between the σ system of the C(α) moiety and the C-O π system of the peptide bond. Maximum and minimum values for each bond distance are found for conformations with the specific bond perpendicular and parallel to the adjacent CONH peptide plane, respectively. On the other hand, the variability of the C-O and C-N distances is related to the strength of the interactions between the lone pair of the N atom and the C-O π* system, which is modulated by the ψ angle. The C-O and C-N distances are related but their trends are not strictly connected to peptide-bond planarity, although a correlation amongst all of these parameters is expected on the basis of the classical resonance model. PMID:26057667

  4. Efficient production of glucose by microwave-assisted acid hydrolysis of cellulose hydrogel.

    PubMed

    Sun, Binzhe; Duan, Lian; Peng, Gege; Li, Xiaoxia; Xu, Aihua

    2015-09-01

    To improve the production of glucose from cellulose, a simple and effective route was developed. This process uses a combination of a step of cellulose dissolution in aqueous NaOH/urea solution and then regeneration with water, followed by an acid hydrolysis step under microwave irradiation. The method is effective to obtain glucose from α-cellulose, microcrystalline cellulose, filter paper, ramie fiber and absorbent cotton. Increased with the acid concentration the glucose yield from hydrogel hydrolysis increased from 0.42% to 44.6% at 160 °C for 10 min. Moreover, the ozone treatment of cellulose in NaOH/urea solution before regeneration significantly enhanced the hydrolysis efficiency with a glucose yield of 59.1%. It is believed that the chains in cellulose hydrogel are relatively free approached, making that the acids easily access the β-glycosidic bonds. PMID:26038330

  5. Ab Initio QM/MM Study of the Ester-hydrolysis Reaction Mechanism in Haloalkane Dehalogenase

    NASA Astrophysics Data System (ADS)

    Zhang, Yiming; Zhou, Yu; Nayak, Saroj; Garcia, Angel

    2006-03-01

    Ab Initio QM/MM calculations are used to investigate the ester-hydrolysis step of dichloroethane hydrolysis catalyzed by haloalkane dehalogenase. Amino acids around the active site (which includes ASP124, HIS289, ASP260, TRP125, TRP175), dichoroethane and water are treated by QM at a level of HF/6-31G(d,p). The remainder of the protein and solvent are treated classically. Two scenarios of hydrolysis mechanism for the alkyl-enzyme intermediate have been considered. In one, the HIS289-catalyzed water oxygen could be incorporated in the carboxylate group of ASP124, leading the cleavage of one of the original carbonyl bonds on ASP124. In the other, the ASP124 and HIS289 as general base, activate water as the nucleophilic agent, which attacks the alkyl carbon in substrate. The reaction paths and potential energy profiles are compared for both mechanisms.

  6. Enzymatic Hydrolysis of Polyester Thin Films: Real-Time Analysis of Film Mass Changes and Dissipation Dynamics.

    PubMed

    Zumstein, Michael Thomas; Kohler, Hans-Peter E; McNeill, Kristopher; Sander, Michael

    2016-01-01

    Cleavage of ester bonds by extracellular microbial hydrolases is considered a key step during the breakdown of biodegradable polyester materials in natural and engineered systems. Here we present a novel analytical approach for simultaneous detection of changes in the masses and rigidities of polyester thin films during enzymatic hydrolysis using a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). In experiments with poly(butylene succinate) (PBS) and the lipase of Rhizopus oryzae (RoL), we detected complete hydrolysis of PBS thin films at pH 5 and 40 °C that proceeded through soft and water-rich film intermediates. Increasing the temperature from 20 to 40 °C resulted in a larger increase of the enzymatic hydrolysis rate of PBS than of nonpolymeric dibutyl adipate. This finding was ascribed to elevated accessibility of ester bonds to the catalytic site of RoL due to increasing polyester chain mobility. When the pH of the solution was changed from 5 to 7, initial hydrolysis rates were little affected, while a softer film intermediate that lead to incomplete film hydrolysis was formed. Hydrolysis dynamics of PBS, poly(butylene adipate), poly(lactic acid), and poly(ethylene terephthalate) in assays with RoL showed distinct differences that we attribute to differences in the polyester structure. PMID:26599203

  7. Yankee bonds

    SciTech Connect

    Delaney, P. )

    1993-10-01

    Yankee and Euromarket bonds may soon find their way into the financing of power projects in Latin America. For developers seeking long-term commitments under build, own, operate, and transfer (BOOT) power projects in Latin America, the benefits are substantial.

  8. How Nature Morphs Peptide Scaffolds into Antibiotics

    PubMed Central

    Nolan, Elizabeth M.; Walsh, Christopher T.

    2010-01-01

    The conventional notion that peptides are poor candidates for orally available drugs because of protease-sensitive peptide bonds, intrinsic hydrophilicity, and ionic charges contrasts with the diversity of antibiotic natural products with peptide-based frameworks that are synthesized and utilized by Nature. Several of these antibiotics, including penicillin and vancomycin, are employed to treat bacterial infections in humans and have been best-selling therapeutics for decades. Others might provide new platforms for the design of novel therapeutics to combat emerging antibiotic-resistant bacterial pathogens. PMID:19058272

  9. Effects of crystallinity on dilute acid hydrolysis of cellulose by cellulose ball-milling study

    SciTech Connect

    Zhao, Haibo; Kwak, Ja Hun; Wang, Yong; Franz, James A.; White, John M.; Holladay, Johnathan E.

    2005-12-23

    The dilute acid (0.05 M H2SO4) hydrolysis at 175°C of samples comprising varying fractions of crystalline (α-form) and amorphous cellulose was studied. The amorphous content, based on XRD and NMR, and then the product (glucose) yield, based on HPLC, increased by as much as a factor of three upon ball milling. These results are interpreted in terms of a model involving mechanical disruption of crystallinity by breaking hydrogen bonds in α-cellulose, opening up the structure and making more β-1,4 glycosidic bonds readily accessible to the dilute acid. In parallel with hydrolysis to form liquid phase products, there are reactions of amorphous cellulose that form solid degradation products.

  10. Resolving Apparent Conflicts between Theoretical and Experimental Models of Phosphate Monoester Hydrolysis

    PubMed Central

    2014-01-01

    Understanding phosphoryl and sulfuryl transfer is central to many biochemical processes. However, despite decades of experimental and computational studies, a consensus concerning the precise mechanistic details of these reactions has yet to be reached. In this work we perform a detailed comparative theoretical study of the hydrolysis of p-nitrophenyl phosphate, methyl phosphate and p-nitrophenyl sulfate, all of which have served as key model systems for understanding phosphoryl and sulfuryl transfer reactions, respectively. We demonstrate the existence of energetically similar but mechanistically distinct possibilities for phosphate monoester hydrolysis. The calculated kinetic isotope effects for p-nitrophenyl phosphate provide a means to discriminate between substrate- and solvent-assisted pathways of phosphate monoester hydrolysis, and show that the solvent-assisted pathway dominates in solution. This preferred mechanism for p-nitrophenyl phosphate hydrolysis is difficult to find computationally due to the limitations of compressing multiple bonding changes onto a 2-dimensional energy surface. This problem is compounded by the need to include implicit solvation to at least microsolvate the system and stabilize the highly charged species. In contrast, methyl phosphate hydrolysis shows a preference for a substrate-assisted mechanism. For p-nitrophenyl sulfate hydrolysis there is only one viable reaction pathway, which is similar to the solvent-assisted pathway for phosphate hydrolysis, and the substrate-assisted pathway is not accessible. Overall, our results provide a unifying mechanistic framework that is consistent with the experimentally measured kinetic isotope effects and reconciles the discrepancies between theoretical and experimental models for these biochemically ubiquitous classes of reaction. PMID:25423607

  11. Hydrolysis of Sulfur Dioxide in Small Clusters of Sulfuric Acid: Mechanistic and Kinetic Study.

    PubMed

    Liu, Jingjing; Fang, Sheng; Wang, Zhixiu; Yi, Wencai; Tao, Fu-Ming; Liu, Jing-Yao

    2015-11-17

    The deposition and hydrolysis reaction of SO2 + H2O in small clusters of sulfuric acid and water are studied by theoretical calculations of the molecular clusters SO2-(H2SO4)n-(H2O)m (m = 1,2; n = 1,2). Sulfuric acid exhibits a dramatic catalytic effect on the hydrolysis reaction of SO2 as it lowers the energy barrier by over 20 kcal/mol. The reaction with monohydrated sulfuric acid (SO2 + H2O + H2SO4 - H2O) has the lowest energy barrier of 3.83 kcal/mol, in which the cluster H2SO4-(H2O)2 forms initially at the entrance channel. The energy barriers for the three hydrolysis reactions are in the order SO2 + (H2SO4)-H2O > SO2 + (H2SO4)2-H2O > SO2 + H2SO4-H2O. Furthermore, sulfurous acid is more strongly bonded to the hydrated sulfuric acid (or dimer) clusters than the corresponding reactant (monohydrated SO2). Consequently, sulfuric acid promotes the hydrolysis of SO2 both kinetically and thermodynamically. Kinetics simulations have been performed to study the importance of these reactions in the reduction of atmospheric SO2. The results will give a new insight on how the pre-existing aerosols catalyze the hydrolysis of SO2, leading to the formation and growth of new particles. PMID:26450714

  12. Solvent influence on cellulose 1,4-β-glycosidic bond cleavage: a molecular dynamics and metadynamics study.

    PubMed

    Loerbroks, Claudia; Boulanger, Eliot; Thiel, Walter

    2015-03-27

    We explore the influence of two solvents, namely water and the ionic liquid 1-ethyl-3-methylimidazolium acetate (EmimAc), on the conformations of two cellulose models (cellobiose and a chain of 40 glucose units) and the solvent impact on glycosidic bond cleavage by acid hydrolysis by using molecular dynamics and metadynamics simulations. We investigate the rotation around the glycosidic bond and ring puckering, as well as the anomeric effect and hydrogen bonds, in order to gauge the effect on the hydrolysis mechanism. We find that EmimAc eases hydrolysis through stronger solvent-cellulose interactions, which break structural and electronic barriers to hydrolysis. Our results indicate that hydrolysis in cellulose chains should start from the ends and not in the centre of the chain, which is less accessible to solvent. PMID:25689773

  13. Continuous steam hydrolysis of tulip poplar

    SciTech Connect

    Fieber, C.A.; Roberts, R.S.; Faass, G.S.; Muzzy, J.D.; Colcord, A.R.; Bery, M.K.

    1982-01-01

    The continuous hydrolysis of poplar chips by steam at 300-350 psi resulted in the separation of hemicellulose (I) cellulose and lignin components. The I fraction was readily depolymerised by steam to acetic acid, furfural, methanol, and xylose.

  14. Antimicrobial peptides.

    PubMed

    Zhang, Ling-Juan; Gallo, Richard L

    2016-01-11

    Antimicrobial peptides and proteins (AMPs) are a diverse class of naturally occurring molecules that are produced as a first line of defense by all multicellular organisms. These proteins can have broad activity to directly kill bacteria, yeasts, fungi, viruses and even cancer cells. Insects and plants primarily deploy AMPs as an antibiotic to protect against potential pathogenic microbes, but microbes also produce AMPs to defend their environmental niche. In higher eukaryotic organisms, AMPs can also be referred to as 'host defense peptides', emphasizing their additional immunomodulatory activities. These activities are diverse, specific to the type of AMP, and include a variety of cytokine and growth factor-like effects that are relevant to normal immune homeostasis. In some instances, the inappropriate expression of AMPs can also induce autoimmune diseases, thus further highlighting the importance of understanding these molecules and their complex activities. This Primer will provide an update of our current understanding of AMPs. PMID:26766224

  15. Towards generation of bioactive peptides from meat industry waste proteins: Generation of peptides using commercial microbial proteases.

    PubMed

    Ryder, Kate; Bekhit, Alaa El-Din; McConnell, Michelle; Carne, Alan

    2016-10-01

    Five commercially available food-grade microbial protease preparations were evaluated for their ability to hydrolyse meat myofibrillar and connective tissue protein extracts to produce bioactive peptides. A bacterial-derived protease (HT) extensively hydrolysed both meat protein extracts, producing peptide hydrolysates with significant in vitro antioxidant and ACE inhibitor activities. The hydrolysates retained bioactivity after simulated gastrointestinal hydrolysis challenge. Gel permeation chromatography sub-fractionation of the crude protein hydrolysates showed that the smaller peptide fractions exhibited the highest antioxidant and ACE inhibitor activities. OFFGEL electrophoresis of the small peptides of both hydrolysates showed that low isoelectric point peptides had antioxidant activity; however, no consistent relationship was observed between isoelectric point and ACE inhibition. Cell-based assays indicated that the hydrolysates present no significant cytotoxicity towards Vero cells. The results indicate that HT protease hydrolysis of meat myofibrillar and connective tissue protein extracts produces bioactive peptides that are non-cytotoxic, should be stable in the gastrointestinal tract and may contain novel bioactive peptide sequences. PMID:27132822

  16. Synthesis of peptide and pseudopeptide amides inhibiting the proliferation of small cell and epithelial types of lung carcinoma cells.

    PubMed

    Nyéki, O; Rill, A; Schon, I; Orosz, A; Schrett, J; Bartha, L; Nagy, J

    1998-12-01

    Small cell lung cancer (SCLC) cell lines produce and secrete various peptide hormones, e.g. bombesin (BN)/gastrin releasing peptide (GRP) like peptides that are proposed to function as their autocrine growth factors. To inhibit the proliferative effect of these hormones we have synthesized short chain BN[7-14]-analogues replacing the C-terminal peptide bond by a methylene-amino (-CH2NH-) unit and introducing D-Phe or D-Ser into position 12. As several substance P (SP) analogues were found to inhibit the growth of SCLC cells, some short chain SP-analogues have been synthesized. (Pseudo)octapeptides were synthesized in solution, by fragment condensation using the DCC/HOPfp method. Fragments and SP-analogues were synthesized stepwise using pentafluorophenyl esters. The resistance to hydrolysis of the reduced peptide bond made permitted exact quantification of the Leupsi(CH2NH)Leu pseudopeptide in hydrolysates. The binding ability of both types of peptides to BN-receptors on Swiss 3T3 mouse fibroblast cells and their antiproliferative effect on NCI-H69 human SCLC cell line have been tested and compared with a short chain SP-antagonist pHOPA-D-Trp-Phe-D-Trp-Leu-Leu-NH2 (R) previously described as a potent inhibitor of SCLC proliferation. While BN-analogues showed weak activity in inhibition of proliferation of SCLC cells, SP-analogues 6: D-MePhe-D-Trp-Phe-D-Trp-Leu(psi)(CH2NH)-Leu-NH2 and 7: D-MePhe-DTrp-Phe-D-Trp-Leu-MPA, in spite of greatly diminished affinity towards the BN-receptor, inhibited SCLC proliferation more effectively than R (6: IC50 = 2 microM, 7: IC50 = 5 microM and R: IC50 = 10 microM). Moreover, 6 inhibited the respiratory activity of SK-MES 1 epithelial type of lung carcinoma cells in proliferating but not in the quiescent state, suggesting that the antiproliferative effect of these compounds is not due to simple cytotoxicity. These short chain analogues of SP might be promising candidates as therapeutic agents in the treatment of SCLC. PMID:9927255

  17. Peptides from milk proteins and their properties.

    PubMed

    Kilara, Arun; Panyam, Dinakar

    2003-01-01

    This review has attempted to study the literature pertaining to peptides derived from milk proteins. Hydrolysis of milk proteins to generate peptides has been practiced for a long time and it was recognized early on in this process that the taste of hydrolyzates might hinder use of these products in food formulations. Modification of protein is necessary to form a more acceptable or utilizable product, to form a product that is less susceptible to deteriorative reactions and to form a product that is of higher nutritionall quality. Modifications may be achieved by a number of chemical and enzymatic means. This review has considered only enzymatic modification of dairy proteins. Modified proteins contain peptides and some of these peptides have been purified and their functionalities have been compared with unmodified proteins. This paper has examined the literature pertaining to improvement in functionality of enzyme-modified proteins. Improvements in solubility, emulsification, foaming and gelation were examined. There is limited information available on the sequence of the peptides necessary to improve the functional characteristics of proteins. Knowing the sequences of desirable functional peptides can lead to genetic alteration of proteins to improve functionality. Addition of synthetic peptides to intact proteins may be another way in which the functionality of proteins can be augmented. Some of the peptides in milk proteins are capable of affecting biological functions of an organism. These effects can be antimicrobial and probiotic, i.e., prevent the growth and proliferation of undesirable and pathogenic organisms, or they may promote the growth of desirable bacteria in the digestive tract of humans and animals. Peptides derived from milk protein have been shown to exert digestive and metabolic effects as well. They may also influence the immune system. These biological effects may play an important role in the development of medical foods that treat or

  18. Method for synthesizing peptides with saccharide linked enzyme polymer conjugates

    DOEpatents

    Callstrom, Matthew R.; Bednarski, Mark D.; Gruber, Patrick R.

    1997-01-01

    A method is disclosed for synthesizing peptides using water soluble enzyme polymer conjugates. The method comprises catalyzing the peptide synthesis with enzyme which has been covalently bonded to a polymer through at least three linkers which linkers have three or more hydroxyl groups. The enzyme is conjugated at lysines or arginines.

  19. Method for synthesizing peptides with saccharide linked enzyme polymer conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-06-17

    A method is disclosed for synthesizing peptides using water soluble enzyme polymer conjugates. The method comprises catalyzing the peptide synthesis with enzyme which has been covalently bonded to a polymer through at least three linkers which linkers have three or more hydroxyl groups. The enzyme is conjugated at lysines or arginines. 19 figs.

  20. Diffusion bonding

    DOEpatents

    Anderson, Robert C.

    1976-06-22

    1. A method for joining beryllium to beryllium by diffusion bonding, comprising the steps of coating at least one surface portion of at least two beryllium pieces with nickel, positioning a coated surface portion in a contiguous relationship with an other surface portion, subjecting the contiguously disposed surface portions to an environment having an atmosphere at a pressure lower than ambient pressure, applying a force upon the beryllium pieces for causing the contiguous surface portions to abut against each other, heating the contiguous surface portions to a maximum temperature less than the melting temperature of the beryllium, substantially uniformly decreasing the applied force while increasing the temperature after attaining a temperature substantially above room temperature, and maintaining a portion of the applied force at a temperature corresponding to about maximum temperature for a duration sufficient to effect the diffusion bond between the contiguous surface portions.

  1. Isolation and characterization of four antibacterial peptides from bovine hemoglobin.

    PubMed

    Nedjar-Arroume, Naima; Dubois-Delval, Véronique; Miloudi, Khalil; Daoud, Rachid; Krier, François; Kouach, Mostafa; Briand, Gilbert; Guillochon, Didier

    2006-09-01

    Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields several intermediate peptide fractions after separation by reversed phase HPLC exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis. From these fractions, four new antibacterial peptides were isolated and analyzed by ESI-MS/MS. Three of these peptides correspond to fragments of the alpha-chain of bovine hemoglobin: alpha107-141, alpha137-141, and alpha133-141, and one peptide to the beta-chain: beta126-145. The minimum inhibitory concentrations (MIC) of these peptides towards the four strains and their hemolytic activity towards bovine erythrocytes were determined. PMID:16730859

  2. Total Chemical Synthesis of a Heterodimeric Interchain Bis-Lactam-Linked Peptide: Application to an Analogue of Human Insulin-Like Peptide 3

    PubMed Central

    Karas, John; Shabanpoor, Fazel; Hossain, Mohammed Akhter; Wade, John D.; Scanlon, Denis B.

    2013-01-01

    Nonreducible cystine isosteres represent important peptide design elements in that they can maintain a near-native tertiary conformation of the peptide while simultaneously extending the in vitro and in vivo half-life of the biomolecule. Examples of these cystine mimics include dicarba, diselenide, thioether, triazole, and lactam bridges. Each has unique physicochemical properties that impact upon the resulting peptide conformation. Each also requires specific conditions for its formation via chemical peptide synthesis protocols. While the preparation of peptides containing two lactam bonds within a peptide is technically possible and reported by others, to date there has been no report of the chemical synthesis of a heterodimeric peptide linked by two lactam bonds. To examine the feasibility of such an assembly, judicious use of a complementary combination of amine and acid protecting groups together with nonfragment-based, total stepwise solid phase peptide synthesis led to the successful preparation of an analogue of the model peptide, insulin-like peptide 3 (INSL3), in which both of the interchain disulfide bonds were replaced with a lactam bond. An analogue containing a single disulfide-substituted interchain lactam bond was also prepared. Both INSL3 analogues retained significant cognate RXFP2 receptor binding affinity. PMID:24288548

  3. Peptide arrays for screening cancer specific peptides.

    PubMed

    Ahmed, Sahar; Mathews, Anu Stella; Byeon, Nara; Lavasanifar, Afsaneh; Kaur, Kamaljit

    2010-09-15

    In this paper, we describe a novel method to screen peptides for specific recognition by cancer cells. Seventy peptides were synthesized on a cellulose membrane in an array format, and a direct method to study the peptide-whole cell interaction was developed. The relative binding affinity of the cells for different peptides with respect to a lead 12-mer p160 peptide, identified by phage display, was evaluated using the CyQUANT fluorescence of the bound cells. Screening allowed identification of at least five new peptides that displayed higher affinity (up to 3-fold) for MDA-MB-435 and MCF-7 human cancer cells compared to the p160 peptide. These peptides showed very little binding to the control (noncancerous) human umbilical vein endothelial cells (HUVECs). Three of these peptides were synthesized separately and labeled with fluorescein isothiocyanate (FITC) to study their uptake and interaction with the cancer and control cells using confocal laser scanning microscopy and flow cytometry. The results confirmed the high and specific affinity of an 11-mer peptide 11 (RGDPAYQGRFL) and a 10-mer peptide 18 (WXEAAYQRFL) for the cancer cells versus HUVECs. Peptide 11 binds different receptors on target cancer cells as its sequence contains multiple recognition motifs, whereas peptide 18 binds mainly to the putative p160 receptor. The peptide array-whole cell binding assay reported here is a complementary method to phage display for further screening and optimization of cancer targeting peptides for cancer therapy and diagnosis. PMID:20799711

  4. Mechanism of Orlistat Hydrolysis by the Thioesterase of Human Fatty Acid Synthase

    PubMed Central

    2015-01-01

    Fatty acid synthase (FASN), the sole protein capable of de novo synthesis of free fatty acids, is overexpressed in a wide variety of human cancers and is associated with poor prognosis and aggressiveness of these cancers. Orlistat, an FDA-approved drug for obesity treatment that inhibits pancreatic lipases in the GI tract, also inhibits the thioesterase (TE) of human FASN. The cocrystal structure of TE with orlistat shows a pseudo TE dimer containing two different forms of orlistat in the active site, an intermediate that is covalently bound to a serine residue (Ser2308) and a hydrolyzed and inactivated product. In this study, we attempted to understand the mechanism of TE-catalyzed orlistat hydrolysis by examining the role of the hexyl tail of the covalently bound orlistat in water activation for hydrolysis using molecular dynamics simulations. We found that the hexyl tail of the covalently bound orlistat undergoes a conformational transition, which is accompanied by destabilization of a hydrogen bond between a hydroxyl moiety of orlistat and the catalytic His2481 of TE that in turn leads to an increased hydrogen bonding between water molecules and His2481 and increased chance for water activation to hydrolyze the covalent bond between orlistat and Ser2308. Thus, the conformation of the hexyl tail of orlistat plays an important role in orlistat hydrolysis. Strategies that stabilize the hexyl tail may lead to the design of more potent irreversible inhibitors that target FASN and block TE activity with greater endurance. PMID:25309810

  5. Effect of ultrasonic pretreatment on kinetics of gelatin hydrolysis by collagenase and its mechanism.

    PubMed

    Yu, Zhi-Long; Zeng, Wei-Cai; Zhang, Wen-Hua; Liao, Xue-Pin; Shi, Bi

    2016-03-01

    Gelatin is a mixture of soluble proteins prepared by partial hydrolysis of native collagen. Gelatin can be enzymatically hydrolyzed to produce bioactive hydrolysates. However, the preparation of gelatin peptide with expected activity is usually a time-consuming process. The production efficiency of gelatin hydrolysates needs to be improved. In present work, effect of ultrasonic pretreatment on kinetic parameters of gelatin hydrolysis by collagenase was investigated based on an established kinetic model. With ultrasonic pretreatment, reaction rate constant and enzyme inactivation constant were increased by 27.5% and 27.8%, respectively. Meanwhile, hydrolysis activation energy and enzyme inactivation energy were reduced by 36.3% and 43.0%, respectively. In order to explore its possible mechanism, influence of sonication on structural properties of gelatin was determined using atomic force microscopy, particle size analyzer, fluorescence spectroscopy, protein solubility test and Fourier transform infrared spectroscopy. Moreover, hydrogen peroxide was used as a positive control for potential sonochemical effect. It was found that reduction of gelatin particle size was mainly caused by physical effect of ultrasound. Increased solubility and variation in β-sheet and random coil elements of gelatin were due to sonochemical effect. Both physical and chemical effects of sonication contributed to the change in α-helix and β-turn structures. The current results suggest that ultrasound can be potentially applied to stimulate the production efficiency of gelatin peptides, mainly due to its effects on modification of protein structures. PMID:26558996

  6. Contribution of hydrogen bonds to protein stability.

    PubMed

    Pace, C Nick; Fu, Hailong; Lee Fryar, Katrina; Landua, John; Trevino, Saul R; Schell, David; Thurlkill, Richard L; Imura, Satoshi; Scholtz, J Martin; Gajiwala, Ketan; Sevcik, Jozef; Urbanikova, Lubica; Myers, Jeffery K; Takano, Kazufumi; Hebert, Eric J; Shirley, Bret A; Grimsley, Gerald R

    2014-05-01

    Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein. PMID:24591301

  7. Contribution of hydrogen bonds to protein stability

    PubMed Central

    Pace, C Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R; Schell, David; Thurlkill, Richard L; Imura, Satoshi; Scholtz, J Martin; Gajiwala, Ketan; Sevcik, Jozef; Urbanikova, Lubica; Myers, Jeffery K; Takano, Kazufumi; Hebert, Eric J; Shirley, Bret A; Grimsley, Gerald R

    2014-01-01

    Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein. PMID:24591301

  8. Contribution of Hydrogen Bonds to Protein Stability

    NASA Astrophysics Data System (ADS)

    Pace, Nick

    2014-03-01

    I will discuss the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(Δ G), for a series of hydrogen bonding mutants in four proteins: villin head piece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A (1.1Å), Y51F(1.5Å), and T95A(1.3Å). The structures are very similar to wild type RNase Sa and the hydrogen bonding partners always form intermolecular hydrogen bonds to water in the mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions: 1) Hydrogen bonds contribute favorably to protein stability. 2) The contribution of hydrogen bonds to protein stability is strongly context dependent. 3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. 4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. 5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.

  9. Hydrolysis theory for cisplatin and its analogues based on density functional studies.

    PubMed

    Zhang, Y; Guo, Z; You, X Z

    2001-09-26

    Hydrolysis of cisplatin, the most widely used anticancer drug in the world, is believed to be the key activation step before the drug reaching its intracellular target DNA. To obtain an accurate hydrolysis theory for this important class of square-planar Pt(II) complexes, three typical reactions, i.e., the first and second hydrolyses of cisplatin and the hydrolysis of [Pt(dien)Cl](+) (dien = diethylenetriamine), were studied at the experimental temperature with the solvent effect using mPW1PW91/SDD from a comprehensive methodological study on the Hartree-Fock (HF) ab initio method, electron correlation methods, pure density functional theory (DFT) methods, and hybrid HF-DFT methods with several basis sets. The true five stationary states in the second-order nucleophilic substitution (S(N)2) pathway for the hydrolysis process, namely, reactant (R) --> intermediate 1 (I1) --> TS --> intermediate 2 (I2) --> product (P) were obtained and characterized theoretically for the first time. The most remarkable structural variations and the associated atomic charge variations in the hydrolysis process were found to occur in the equatorial plane of the five-coordinate trigonal-bipyramidal (TBP)-like structures of I1, TS, and I2. The reaction with the TS structure of smaller L-M-E angle and more lengthened M-L and M-E bonds was found to have a smaller Gibbs free energy change and accordingly the better hydrolysis yield. It is found that the sum of the three concentric angles in the TBP's equator is near 360 degrees in I1 and I2 and is almost 360 degrees in TS in each reaction. The associated energy profiles again demonstrated a typical S(N)2 reaction curve. The computed forward and backward reaction enthalpy (Delta H(++)) and reaction entropy (Delta S(++)) in the rate-determining step I1 --> TS --> I2 are in good agreement with the experiments. Natural bonding orbital population analysis shows that the charge-separating extent follows the same order of Delta G in studied

  10. Glutamate carboxypeptidase II is not an amyloid peptide-degrading enzyme.

    PubMed

    Alt, Jesse; Stathis, Marigo; Rojas, Camilo; Slusher, Barbara

    2013-07-01

    Glutamate carboxypeptidase II (GCPII) is an exopeptidase that catalyzes the hydrolysis of N-acetylated aspartate-glutamate (NAAG) to N-acetyl aspartate (NAA) and glutamate. Consequently, GCPII inhibition has been of interest for the treatment of central and peripheral nervous system diseases associated with excess glutamate. Recently, it was reported that GCPII can also serve as an endopeptidase cleaving amyloid β (Aβ) peptides and that its inhibition could increase the risk of Alzheimer's disease by increasing brain Aβ levels. This study aimed to corroborate and extend these new findings. We incubated Aβ peptides (20 μM) with human recombinant GCPII (300 ng/ml) and monitored the appearance of degradation products by mass spectrometry. Aβ peptides remained intact after 18 h incubation with GCPII. Under the same experimental conditions, Aβ1-40 (20 μM) was incubated with neprilysin (300 ng/ml), an endopeptidase known to hydrolyze Aβ1-40 and the expected cleavage products were observed. GCPII was confirmed active by catalyzing the complete hydrolysis of NAAG (100 μM). We also studied the hydrolysis of [(3)H]-NAAG (30 nM) catalyzed by GCPII (40 pM) in the presence of Aβ peptides (picomolar to micromolar range). The addition of Aβ peptides did not alter [(3)H]-NAAG hydrolysis. We conclude that GCPII is not an amyloid peptide-degrading enzyme. PMID:23525278

  11. Pi Bond Orders and Bond Lengths

    ERIC Educational Resources Information Center

    Herndon, William C.; Parkanyi, Cyril

    1976-01-01

    Discusses three methods of correlating bond orders and bond lengths in unsaturated hydrocarbons: the Pauling theory, the Huckel molecular orbital technique, and self-consistent-field techniques. (MLH)

  12. Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass

    SciTech Connect

    Inoue, Hiroyuki; Decker, Stephen R.; Taylor, Larry E.; Yano, Shinichi; Sawayama, Shigeki

    2014-10-09

    Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown. Results: Six cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysis of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan. In Conclusion: Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and

  13. Detection of trans–cis flips and peptide-plane flips in protein structures

    SciTech Connect

    Touw, Wouter G.; Joosten, Robbie P.; Vriend, Gert

    2015-07-28

    A method is presented to detect peptide bonds that need either a trans–cis flip or a peptide-plane flip. A coordinate-based method is presented to detect peptide bonds that need correction either by a peptide-plane flip or by a trans–cis inversion of the peptide bond. When applied to the whole Protein Data Bank, the method predicts 4617 trans–cis flips and many thousands of hitherto unknown peptide-plane flips. A few examples are highlighted for which a correction of the peptide-plane geometry leads to a correction of the understanding of the structure–function relation. All data, including 1088 manually validated cases, are freely available and the method is available from a web server, a web-service interface and through WHAT-CHECK.

  14. Strength of Multiple Parallel Biological Bonds

    SciTech Connect

    Sulchek, T A; Friddle, R W; Noy, A

    2005-12-07

    Multivalent interactions play a critical role in a variety of biological processes on both molecular and cellular levels. We have used molecular force spectroscopy to investigate the strength of multiple parallel peptide-antibody bonds using a system that allowed us to determine the rupture forces and the number of ruptured bonds independently. In our experiments the interacting molecules were attached to the surfaces of the probe and sample of the atomic force microscope with flexible polymer tethers, and unique mechanical signature of the tethers determined the number of ruptured bonds. We show that the rupture forces increase with the number of interacting molecules and that the measured forces obey the predictions of a Markovian model for the strength of multiple parallel bonds. We also discuss the implications of our results to the interpretation of force spectroscopy measurements in multiple bond systems.

  15. Confinement-dependent friction in peptide bundles.

    PubMed

    Erbaş, Aykut; Netz, Roland R

    2013-03-19

    Friction within globular proteins or between adhering macromolecules crucially determines the kinetics of protein folding, the formation, and the relaxation of self-assembled molecular systems. One fundamental question is how these friction effects depend on the local environment and in particular on the presence of water. In this model study, we use fully atomistic MD simulations with explicit water to obtain friction forces as a single polyglycine peptide chain is pulled out of a bundle of k adhering parallel polyglycine peptide chains. The whole system is periodically replicated along the peptide axes, so a stationary state at prescribed mean sliding velocity V is achieved. The aggregation number is varied between k = 2 (two peptide chains adhering to each other with plenty of water present at the adhesion sites) and k = 7 (one peptide chain pulled out from a close-packed cylindrical array of six neighboring peptide chains with no water inside the bundle). The friction coefficient per hydrogen bond, extrapolated to the viscous limit of vanishing pulling velocity V → 0, exhibits an increase by five orders of magnitude when going from k = 2 to k = 7. This dramatic confinement-induced friction enhancement we argue to be due to a combination of water depletion and increased hydrogen-bond cooperativity. PMID:23528088

  16. Review: Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-07-16

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  17. C-Peptide Test

    MedlinePlus

    ... C-peptide is a useful marker of insulin production. The following are some purposes of C-peptide ... it nearly impossible to directly evaluate endogenous insulin production. In these cases, C-peptide measurement is a ...

  18. Cyclic Peptides Made by Linking Synthetic and Genetically Encoded Fragments.

    PubMed

    Palei, Shubhendu; Mootz, Henning D

    2016-03-01

    Cyclic peptides can be highly valuable as bioactive molecules, both for biomedical applications and in basic research. We introduce a new fragment-based approach to access cyclic peptide structures in which one fragment is of synthetic origin and the other is genetically encoded. The synthetic peptide, which can contain one or more non-proteinogenic building blocks, is coupled to the recombinantly expressed peptide through two bonds, one formed by protein trans-splicing with a split intein and the other by oxime ligation. Semisynthetic macrocycles were obtained with high efficiency for various sequences and ring sizes; they can be prepared in quantities sufficient for initial bioactivity tests. We also prepared lipidated and d-amino-acid-containing peptides that were inspired by the peptide antibiotic daptomycin. Such structures are not accessible by other methods that harness the power of simple genetic diversification in the DNA-encoded part of the peptide. PMID:26691013

  19. Bonded Lubricants

    NASA Technical Reports Server (NTRS)

    1977-01-01

    Another spinoff to the food processing industry involves a dry lubricant developed by General Magnaplate Corp. of Linden, N.J. Used in such spacecraft as Apollo, Skylab and Viking, the lubricant is a coating bonded to metal surfaces providing permanent lubrication and corrosion resistance. The coating lengthens equipment life and permits machinery to be operated at greater speed, thus increasing productivity and reducing costs. Bonded lubricants are used in scores of commercia1 applications. They have proved particularly valuable to food processing firms because, while increasing production efficiency, they also help meet the stringent USDA sanitation codes for food-handling equipment. For example, a cookie manufacturer plagued production interruptions because sticky batter was clogging the cookie molds had the brass molds coated to solve the problem. Similarly, a pasta producer faced USDA action on a sanitation violation because dough was clinging to an automatic ravioli-forming machine; use of the anti-stick coating on the steel forming plates solved the dual problem of sanitation deficiency and production line downtime.

  20. Mechanical tuning of elastomers via peptide secondary structure

    NASA Astrophysics Data System (ADS)

    Wanasekara, Nandula; Johnson, J. Casey; Korley, Lashanda T. J.

    2014-03-01

    Nature utilizes an array of design tools for engineering materials with multiple functions and tunable mechanical properties. The precise control of hierarchical structure, self-assembly, and secondary structure is essential to achieve the desired properties in bio-inspired materials design. We have developed a series of peptidic-poyurea hybrids to determine the effects of peptide secondary structure and hydrogen bonding arrangement on morphology, thermal and mechanical properties. These materials were fabricated by incorporating peptide segments containing either poly(β-benzyl-L-aspartate) or poly(ɛ-carbobenzyloxy-L-lysine) into non-chain extended polyureas to form either β-sheets or α-helix conformations based on peptide length. Infrared analysis proved the retention of peptide secondary structure when incorporated into peptidic-polyureas. The polymers containing β-sheet forming peptide blocks exhibited higher modulus and toughness due to intermolecular H-bonding. Additionally, higher peptide weight fractions lead to higher plateau moduli due to a transition of continuous domain morphology from a soft segment continuous to a fibrous and interconnected stiffer peptide domain. All the polymers exhibited microphase separated morphology with nanofibrous or ribbon-like structures. It is observed that fiber aspect ratio and percolation were influenced by the peptide secondary structure and the weight fraction.

  1. Is aspartate 52 essential for catalysis by chicken egg white lysozyme? The role of natural substrate-assisted hydrolysis

    SciTech Connect

    Matsumura, Ichiro; Kirsch, J.F.

    1996-02-13

    The chicken and goose egg white lysozymes (ChEWL and GoEWL) are homologues, but differ in substrate specificity. ChEWL catalyzes the hydrolysis of the glycosidic bonds of bacterial peptidoglycans and chitin-derived substrates, while GoEWL is specific for bacterial peptidoglycans. The active-site aspartate 52 residue of ChEWL, which is postulated to stabilize the oxocarbenium ion intermediate, has no counterpart in GoEWL. The substrate specificity of the D52A ChEWL mutant was compared with those of wild-type ChEWL and GoEWL. D52A ChEWL retains approximately 4% of the wild-type catalytic activity in reactions with three different bacterial cell suspensions. Asp52 therefore is not essential to the catalytic mechanism, accounting for only a 2 kcal/mol decrease in AG. The function of Asp52 in D52A ChEWL- and GoEWL-catalyzed cleavage of (carboxymethyl)chitin may be partially fulfilled by an appropriately positioned carboxyl group on the substrate (substrate-assisted catalysis). D52A ChEWL and GoEWL, unlike wild-type ChEWL, exhibit biphasic kinetics in the clearing of Micrococcus luteus cell suspensions, suggesting preferences for subsets of the linkages in the M. luteus peptidoglycan. These subsets do not exist in the peptidoglycans of Escherichia coli or Sarcina lutea, since neither D52A ChEWL nor GoEWL exhibits initial bursts in reactions with suspensions of these bacteria. We propose that substrate-assisted catalysis occurs in reactions of D52A ChEWL and GoEWL with M. luteus peptidoglycans, with the glycine carboxyl group of uncross-linked peptides attached to N-acetylmuramic acid partially substituting the function of the missing Asp52. 52 refs., 6 figs., 1 tab.

  2. Basics of Fidelity Bonding.

    ERIC Educational Resources Information Center

    Kahn, Steven P.

    Fidelity bonds are important for an agency to hold to protect itself against any financial loss that can result from dishonest acts by its employees. Three types of fidelity bonds are available to an agency: (1) public official bonds; (2) dishonesty bonds; and (3) faithful performance bonds. Public official bonds are required by state law to be…

  3. Peptide synthesis in early earth hydrothermal systems

    USGS Publications Warehouse

    Lemke, K.H.; Rosenbauer, R.J.; Bird, D.K.

    2009-01-01

    We report here results from experiments and thermodynamic calculations that demonstrate a rapid, temperature-enhanced synthesis of oligopeptides from the condensation of aqueous glycine. Experiments were conducted in custom-made hydrothermal reactors, and organic compounds were characterized with ultraviolet-visible procedures. A comparison of peptide yields at 260??C with those obtained at more moderate temperatures (160??C) gives evidence of a significant (13 kJ ?? mol-1) exergonic shift. In contrast to previous hydrothermal studies, we demonstrate that peptide synthesis is favored in hydrothermal fluids and that rates of peptide hydrolysis are controlled by the stability of the parent amino acid, with a critical dependence on reactor surface composition. From our study, we predict that rapid recycling of product peptides from cool into near-supercritical fluids in mid-ocean ridge hydrothermal systems will enhance peptide chain elongation. It is anticipated that the abundant hydrothermal systems on early Earth could have provided a substantial source of biomolecules required for the origin of life. Astrobiology 9, 141-146. ?? 2009 Mary Ann Liebert, Inc. 2009.

  4. Antigenic mimicry of natural L-peptides with retro-inverso-peptidomimetics.

    PubMed Central

    Guichard, G; Benkirane, N; Zeder-Lutz, G; van Regenmortel, M H; Briand, J P; Muller, S

    1994-01-01

    Three analogues of the model peptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 were synthesized, and their antigenicity, immunogenicity, and resistance to trypsin were compared to those of the natural L-peptide. The three analogues correspond to the D-enantiomer, containing only D-residues, and two retro-peptides containing NH-CO bonds instead of natural peptide bonds. The chirality of each residue was maintained in the retro-peptide and inverted in the retro-inverso-peptide. Antibodies to the four peptide analogues were produced by injecting BALB/c mice with peptides covalently coupled to small unilamellar liposomes containing monophosphoryl lipid A. Each of the four peptide analogues induced IgG antibodies of various subclasses. The IgG3 antibodies reacted similarly with the four analogues, whereas antibodies of the IgG1, IgG2a, and IgG2b isotypes showed strong conformational preferences for certain peptides. The retro-inverso-peptide IRGERA mimicked the structure and antigenic activity of the natural L-peptide but not of the D- and retro-peptides, whereas the retro-peptide IRGERA mimicked the D-peptide but not the L- and retro-inverso-peptides. The equilibrium affinity constants (Ka) of three monoclonal antibodies generated against the L- and D-peptides with respect to the four peptide analogues were measured in a biosensor system. Large differences in Ka values were observed when each monoclonal antibody was tested with respect to the four peptides. The use of retro-inverso-peptides to replace natural L-peptides is likely to find many applications in immunodiagnosis and as potential synthetic vaccines. Images PMID:7937888

  5. Disulphide bond assignment in human tissue inhibitor of metalloproteinases (TIMP).

    PubMed Central

    Williamson, R A; Marston, F A; Angal, S; Koklitis, P; Panico, M; Morris, H R; Carne, A F; Smith, B J; Harris, T J; Freedman, R B

    1990-01-01

    Disulphide bonds in human recombinant tissue inhibitor of metalloproteinases (TIMP) were assigned by resolving proteolytic digests of TIMP on reverse-phase h.p.l.c. and sequencing those peaks judged to contain disulphide bonds by virtue of a change in retention time on reduction. This procedure allowed the direct assignment of Cys-145-Cys-166 and the isolation of two other peptides containing two disulphide bonds each. Further peptide cleavage in conjunction with fast-atom-bombardment m.s. analysis permitted the assignments Cys-1-Cys-70, Cys-3-Cys-99, Cys-13-Cys-124 and Cys-127-Cys-174 from these peptides. The sixth bond Cys-132-Cys-137 was assigned by inference, as the native protein has no detectable free thiol groups. Images Fig. 1. PMID:2163605

  6. Peptide T, a novel bradykinin potentiator isolated from Tityus serrulatus scorpion venom.

    PubMed

    Ferreira, L A; Alves, E W; Henriques, O B

    1993-08-01

    A bradykinin-potentiating peptide was isolated and characterized from venom of the scorpion Tityus serrulatus by chromatographic techniques followed by biological assays. The complete amino acid sequence (13 residues) of peptide is presented. The peptide potentiated the contractile activity of bradykinin on the isolated guinea-pig ileum, and inhibited the hydrolysis of bradykinin by angiotensin-converting enzyme from B. jararaca plasma and the conversion of angiotensin I to angiotensin II by kininase II from guinea-pig ileum tissue. The peptide also increased the depressor effect of bradykinin on arterial blood pressure in the anaesthetized rat. PMID:8212046

  7. Investigation of purification process stresses on erythropoietin peptide mapping profile

    PubMed Central

    Sepahi, Mina; Kaghazian, Hooman; Hadadian, Shahin; Norouzian, Dariush

    2015-01-01

    Background: Full compliance of recombinant protein peptide mapping chromatogram with the standard reference material, is one of the most basic quality control tests of biopharmaceuticals. Changing a single amino acid substitution or side chain diversity for a given peptide changes protein hydrophobicity and causes peak shape or retention time alteration in a peptide mapping assay. In this work, the effect of different stresses during the recombinant erythropoietin (EPO) purification process, including pH 4, pH 5, and room temperature were checked on product peptide mapping results. Materials and Methods: Cell culture harvest was purified under stress by different chromatographic techniques consisting of gel filtration, anionic ion exchange, concentration by ultrafiltration, and high resolution size exclusion chromatography. To induce more pH stresses, the purified EPO was exposed to pH stress 4 and 5 by exchanging buffer by a 10 KDa dialysis sac overnight. The effects of temperature and partial deglycosylation (acid hydrolysis) on purified EPO were also studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and peptide mapping analysis. Removal of sialic acid by mild hydrolysis was performed by exposure to two molar acetic acid at 80°C for 3 h. Results: No significant effect was observed between intact and stressed erythropoietin peptide mapping profiles and SDS-PAGE results. To validate the sensibility of the technique, erythropoietin was partially acid hydrolyzed and significant changes in the chromatographic peptide map of the intact form and a reduction on its molecular weight were detected, which indicates some partial deglycosylation. Conclusions: Purification process does not alter the peptide mapping profile and purification process stresses are not the cause of peptide mapping noncompliance. PMID:26261816

  8. Camptothecin-catalyzed phospholipid hydrolysis in liposomes.

    PubMed

    Saetern, Ann Mari; Skar, Merete; Braaten, Asmund; Brandl, Martin

    2005-01-01

    Hydrolysis of phospholipid (PL) within camptothecin (CPT)-containing liposomes was studied systematically, after elevated lyso-phosphatidylcholine (LPC)-concentrations in pH 5, CPT-containing liposomes (22.1+/-0.9 mol%) relative to control-liposomes (7.3+/-0.5 mol%) occasionally had been observed after four months storage in fridge. Liposomes were prepared by dispersing freeze-dried PL/CPT mixtures in 25 mM phosphate buffered saline (PBS) of varying pH (5.0-7.8) and CPT concentrations (0, 3 and 6 mM). PL-hydrolysis was monitored by HPTLC, quantifying LPC. In an accelerated stability study (60 degrees C), a catalytic effect of CPT on PL-hydrolysis was observed after 40 h, but not up to 30 h of incubation. The pH profile of the hydrolysis indicated a stability optimum at pH 6.0 for the liposomes independent of CPT. The equilibrium point between the more active lactone- and the carboxylate-form of CPT was found to be pH 6.8. As a compromise, pH 6.0 was chosen, assuring >85% CPT to be present in the lactone form. At this pH, both control- and CPT-liposomes showed only minor hydrolysis after autoclaving (121 degrees C, 15 min). Storage at room temperature and in fridge (2 months), as well as accelerated ageing (70 degrees C, 25 h), gave a significant elevation of LPC content in CPT-liposomes relative to control-liposomes. This study demonstrates a catalytic effect of CPT on PL-hydrolysis, the onset of which seems to require a certain threshold level of hydrolytic degradation. PMID:15607259

  9. Chemical Kinetic Analysis of Thermal Decay of Rhodopsin Reveals Unusual Energetics of Thermal Isomerization and Hydrolysis of Schiff Base*

    PubMed Central

    Liu, Jian; Liu, Monica Yun; Fu, Li; Zhu, Gefei Alex; Yan, Elsa C. Y.

    2011-01-01

    The thermal properties of rhodopsin, which set the threshold of our vision, have long been investigated, but the chemical kinetics of the thermal decay of rhodopsin has not been revealed in detail. To understand thermal decay quantitatively, we propose a kinetic model consisting of two pathways: 1) thermal isomerization of 11-cis-retinal followed by hydrolysis of Schiff base (SB) and 2) hydrolysis of SB in dark state rhodopsin followed by opsin-catalyzed isomerization of free 11-cis-retinal. We solve the kinetic model mathematically and use it to analyze kinetic data from four experiments that we designed to assay thermal decay, isomerization, hydrolysis of SB using dark state rhodopsin, and hydrolysis of SB using photoactivated rhodopsin. We apply the model to WT rhodopsin and E181Q and S186A mutants at 55 °C, as well as WT rhodopsin in H2O and D2O at 59 °C. The results show that the hydrogen-bonding network strongly restrains thermal isomerization but is less important in opsin and activated rhodopsin. Furthermore, the ability to obtain individual rate constants allows comparison of thermal processes under various conditions. Our kinetic model and experiments reveal two unusual energetic properties: the steep temperature dependence of the rates of thermal isomerization and SB hydrolysis in the dark state and a strong deuterium isotope effect on dark state SB hydrolysis. These findings can be applied to study pathogenic rhodopsin mutants and other visual pigments. PMID:21921035

  10. Antimicrobial Lactoferrin Peptides: The Hidden Players in the Protective Function of a Multifunctional Protein

    PubMed Central

    Sinha, Mau; Kaushik, Sanket; Kaur, Punit; Singh, Tej P.

    2013-01-01

    Lactoferrin is a multifunctional, iron-binding glycoprotein which displays a wide array of modes of action to execute its primary antimicrobial function. It contains various antimicrobial peptides which are released upon its hydrolysis by proteases. These peptides display a similarity with the antimicrobial cationic peptides found in nature. In the current scenario of increasing resistance to antibiotics, there is a need for the discovery of novel antimicrobial drugs. In this context, the structural and functional perspectives on some of the antimicrobial peptides found in N-lobe of lactoferrin have been reviewed. This paper provides the comparison of lactoferrin peptides with other antimicrobial peptides found in nature as well as interspecies comparison of the structural properties of these peptides within the native lactoferrin. PMID:23554820

  11. Atomic scale insights into urea-peptide interactions in solution.

    PubMed

    Steinke, Nicola; Gillams, Richard J; Pardo, Luis Carlos; Lorenz, Christian D; McLain, Sylvia E

    2016-02-01

    The mechanism by which proteins are denatured by urea is still not well understood, especially on the atomic scale where these interactions occur in vivo. In this study, the structure of the peptide GPG has been investigated in aqueous urea solutions in order to understand the combination of roles that both urea and water play in protein unfolding. Using a combination of neutron diffraction enhanced by isotopic substitution and computer simulations, it was found, in opposition with previous simulations studies, that urea is preferred over water around polar and charged portions of the peptides. Further, it appears that while urea directly replaces water around the nitrogen groups on GPG that urea and water occupy different positions around the peptide bond carbonyl groups. This suggests that urea may in fact weaken the peptide bond, disrupting the peptide backbone, thus ultimately causing denaturation. PMID:26764567

  12. Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass

    DOE PAGESBeta

    Inoue, Hiroyuki; Decker, Stephen R.; Taylor, Larry E.; Yano, Shinichi; Sawayama, Shigeki

    2014-10-09

    Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown. Results: Six cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysismore » of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan. In Conclusion: Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A

  13. Housefly larvae hydrolysate: orthogonal optimization of hydrolysis, antioxidant activity, amino acid composition and functional properties

    PubMed Central

    2013-01-01

    Background Antioxidant, one of the most important food additives, is widely used in food industry. At present, antioxidant is mostly produced by chemical synthesis, which would accumulate to be pathogenic. Therefore, a great interest has been developed to identify and use natural antioxidants. It was showed that there are a lot of antioxidative peptides in protein hydrolysates, possessing strong capacity of inhibiting peroxidation of macro-biomolecular and scavenging free redicals in vivo. Enzymatic hydrolysis used for preparation of antioxidative peptides is a new hot-spot in the field of natural antioxidants. It reacts under mild conditions, with accurate site-specific degradation, good repeatability and few damages to biological activity of protein. Substrates for enzymatic hydrolysis are usually plants and aqua-animals. Insects are also gaining attention because of their rich protein and resource. Antioxidative peptides are potential to be exploited as new natural antioxidant and functional food. There is a huge potential market in medical and cosmetic field as well. Result Protein hydrolysate with antioxidant activity was prepared from housefly larvae, by a two-step hydrolysis. Through orthogonal optimization of the hydrolysis conditions, the degree of hydrolysis was determined to be approximately 60%. Fractionated hydrolysate at 25 mg/mL, 2.5 mg/mL and 1 mg/mL exhibited approximately 50%, 60% and 50% of scavenging capacity on superoxide radicals, 1, 1-Diphenyl-2-picrylhydrazyl radicals and hydroxyl radicals, respectively. Hydrolysate did not exhibit substantial ion chelation. Using a linoneic peroxidation system, the inhibition activity of hydrolysate at 20 mg/mL was close to that of 20 μg/mL tertiary butylhydroquinone, suggesting a potential application of hydrolysate in the oil industry as an efficient antioxidant. The lyophilized hydrolysate presented almost 100% solubility at pH 3-pH 9, and maintained nearly 100% activity at pH 5-pH 8 at 0

  14. A density functional theory model of mechanically activated silyl ester hydrolysis

    SciTech Connect

    Pill, Michael F.; Schmidt, Sebastian W.; Beyer, Martin K.; Clausen-Schaumann, Hauke; Kersch, Alfred

    2014-01-28

    To elucidate the mechanism of the mechanically activated dissociation of chemical bonds between carboxymethylated amylose (CMA) and silane functionalized silicon dioxide, we have investigated the dissociation kinetics of the bonds connecting CMA to silicon oxide surfaces with density functional calculations including the effects of force, solvent polarizability, and pH. We have determined the activation energies, the pre-exponential factors, and the reaction rate constants of candidate reactions. The weakest bond was found to be the silyl ester bond between the silicon and the alkoxy oxygen atom. Under acidic conditions, spontaneous proton addition occurs close to the silyl ester such that neutral reactions become insignificant. Upon proton addition at the most favored position, the activation energy for bond hydrolysis becomes 31 kJ mol{sup −1}, which agrees very well with experimental observation. Heterolytic bond scission in the protonated molecule has a much higher activation energy. The experimentally observed bi-exponential rupture kinetics can be explained by different side groups attached to the silicon atom of the silyl ester. The fact that different side groups lead to different dissociation kinetics provides an opportunity to deliberately modify and tune the kinetic parameters of mechanically activated bond dissociation of silyl esters.

  15. Chain Length and Grafting Density Dependent Enhancement in the Hydrolysis of Ester-Linked Polymer Brushes.

    PubMed

    Melzak, Kathryn A; Yu, Kai; Bo, Deng; Kizhakkedathu, Jayachandran N; Toca-Herrera, José L

    2015-06-16

    Poly(N,N-dimethylacrylamide) (PDMA) brushes with different grafting density and chain length were grown from an ester group-containing initiator using surface-initiated polymerization. Hydrolysis of the PDMA chains from the surface was monitored by measuring thickness of the polymer layer by ellipsometry and extension length by atomic force microscopy. It was found that the initial rate of cleavage of one end-tethered PDMA chains was dependent on the grafting density and chain length; the hydrolysis rate was faster for high grafting density brushes and brushes with higher molecular weights. Additionally, the rate of cleavage of polymer chains during a given experiment changed by up to 1 order of magnitude as the reaction progressed, with a distinct transition to a lower rate as the grafting density decreased. Also, polymer chains undergo selective cleavage, with longer chains in a polydisperse brush being preferentially cleaved at one stage of the hydrolysis reaction. We suggest that the enhanced initial hydrolysis rates seen at high grafting densities and high chain lengths are due to mechanical activation of the ester bond connecting the polymer chains to the surface in association with high lateral pressure within the brush. These results have implications for the preparation of polymers brushes, their stability under harsh conditions, and the analysis of polymer brushes from partial hydrolysates. PMID:26010390

  16. Optimization of Glutamine Peptide Production from Soybean Meal and Analysis of Molecular Weight Distribution of Hydrolysates

    PubMed Central

    Xie, Yanli; Liang, Xinhong; Wei, Min; Zhao, Wenhong; He, Baoshan; Lu, Qiyu; Huo, Quangong; Ma, Chengye

    2012-01-01

    The process parameters of enzymatic hydrolysis and molecular weight distribution of glutamine (Gln) peptides from soybean meal were investigated. The Protamex® hydrolysis pH of 6.10, temperature of 56.78 °C, enzyme to substrate ratio (E/S) of 1.90 and hydrolysis time of 10.72 h were found to be the optimal conditions by response surface methodology (RSM) for a maximal degree of hydrolysis (DH) value of 16.63% and Gln peptides content at 5.95 mmol/L. The soybean meal was hydrolyzed by a combination of Protamex® and trypsinase so that DH and Gln peptides would reach 22.02% and 6.05 mmol/mL, respectively. The results of size exclusion chromatography indicated that the relative proportion of the molecular weight <1000 Da fraction increased with DH values from 6.76%, 11.13%, 17.89% to 22.02%, most notably the 132–500 Da fractions of hydrolysates were 42.14%, 46.57%, 58.44% and 69.65%. High DH values did not lead to high Gln peptides content of the hydrolysate but to the low molecular weight Gln peptides. PMID:22837706

  17. Synthesis of Peptide Substrates for Mammalian Thioredoxin Reductase†

    PubMed Central

    Flemer, Stevenson; Lacey, Brian M.; Hondal, Robert J.

    2013-01-01

    Mammalian thioredoxin reductase catalyzes the reduction of the redox-active disulfide bond of thioredoxin and is similar in structure and mechanism to glutathione reductase except for a C-terminal 16 amino acid extension containing a rare vicinal selenylsulfide bond. This vicinal selenylsulfide bond is essentially a substrate for the enzyme’s N-terminal redox center. Here we report the synthesis of peptide substrates for the truncated enzyme missing the C-terminal redox center. We developed a procedure for the synthesis of peptides containing cyclic vicinal disulfide/selenylsulfide bonds as well as their corresponding acyclic heterodimers. Vicinal disulfide bonds form 8-membered ring structures and are difficult to synthesize due to their propensity to dimerize during oxidation. Our procedure makes use of two key improvements for on-resin disulfide bond formation presented previously by Galande and coworkers [Galande AK, Weissleder R, Tung C-H. An effective method of on-resin disulfide bond formation in peptides. J. Comb. Chem. 2005; 7: 174–177.]. First, the addition of an amine base to the deprotection solution allows for complete removal of the StBu group, allowing it to be replaced with a 5-Npys group. The second enhancement is the direct use of a Cys(Mob) or Sec(Mob) derivative as the nucleophilic partner instead of utilizing a naked sulfhydryl or selenol. These improvements result in the formation of a vicinal disulfide (or selenylsulfide) bond in high purity and yield. A direct comparison with the Galande procedure is presented. We also present a novel strategy for the formation of an acyclic, interchain selenylsulfide linked peptide (linking H-PTVTGC-OH and H-UG-OH). Cysteine analogs of the cyclic and acyclic peptides were also synthesized. The results show that the ring structure contributes a factor of 52 to the rate, but the presence of selenium in the peptide is more important to catalysis than the presence of the ring. PMID:18035847

  18. Enhanced rigid-bond restraints

    SciTech Connect

    Thorn, Andrea; Dittrich, Birger; Sheldrick, George M.

    2012-07-01

    An extension is proposed to the rigid-bond description of atomic thermal motion in crystals. The rigid-bond model [Hirshfeld (1976 ▶). Acta Cryst. A32, 239–244] states that the mean-square displacements of two atoms are equal in the direction of the bond joining them. This criterion is widely used for verification (as intended by Hirshfeld) and also as a restraint in structure refinement as suggested by Rollett [Crystallographic Computing (1970 ▶), edited by F. R. Ahmed et al., pp. 167–181. Copenhagen: Munksgaard]. By reformulating this condition, so that the relative motion of the two atoms is required to be perpendicular to the bond, the number of restraints that can be applied per anisotropic atom is increased from about one to about three. Application of this condition to 1,3-distances in addition to the 1,2-distances means that on average just over six restraints can be applied to the six anisotropic displacement parameters of each atom. This concept is tested against very high resolution data of a small peptide and employed as a restraint for protein refinement at more modest resolution (e.g. 1.7 Å)

  19. Phosphatase Hydrolysis of Soil Organic Phosphorus Fractions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant available inorganic phosphorus (Pi) is usually limited in highly weathered Ultisols. The high Fe, Al, and Mn contents in these soils enhance Pi retention and fixation. The metals are also known to form complexes with organic phosphorus (Po) compounds. Hydrolysis of Po compounds is needed for P...

  20. Phosphatase hydrolysis of organic phosphorus compounds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphatases are diverse groups of enzymes that deserve special attention because of the significant roles they play in mineralizing organic phosphorus (P) into inorganic available form. For getting more insight on the enzymatically hydrolysis of organic P, in this work, we compared the catalytic pa...

  1. Thioglycoside hydrolysis catalyzed by {beta}-glucosidase

    SciTech Connect

    Shen Hong; Byers, Larry D.

    2007-10-26

    Sweet almond {beta}-glucosidase (EC 3.2.1.21) has been shown to have significant thioglycohydrolase activity. While the K{sub m} values for the S- and O-glycosides are similar, the k{sub cat} values are about 1000-times lower for the S-glycosides. Remarkably, the pH-profile for k{sub cat}/K{sub m} for hydrolysis of p-nitrophenyl thioglucoside (pNPSG) shows the identical dependence on a deprotonated carboxylate (pK{sub a} 4.5) and a protonated group (pK{sub a} 6.7) as does the pH-profile for hydrolysis of the corresponding O-glycoside. Not surprisingly, in spite of the requirement for the presence of this protonated group in catalytically active {beta}-glucosidase, thioglucoside hydrolysis does not involve general acid catalysis. There is no solvent kinetic isotope effect on the enzyme-catalyzed hydrolysis of pNPSG.

  2. Optimization of dilute acid hydrolysis of Enteromorpha

    NASA Astrophysics Data System (ADS)

    Feng, Dawei; Liu, Haiyan; Li, Fuchao; Jiang, Peng; Qin, Song

    2011-11-01

    Acid hydrolysis is a simple and direct way to hydrolyze polysaccharides in biomass into fermentable sugars. To produce fermentable sugars effectively and economically for fuel ethanol, we have investigated the hydrolysis of Enteromorpha using acids that are typically used to hydrolyze biomass: H2SO4, HCl, H3PO4 and C4H4O4 (maleic acid). 5%(w/w) Enteromorpha biomass was treated for different times (30, 60, and 90 min) and with different acid concentrations (0.6, 1.0, 1.4, 1.8, and 2.2%, w/w) at 121°C. H2SO4 was the most effective acid in this experiment. We then analyzed the hydrolysis process in H2SO4 in detail using high performance liquid chromatography. At a sulfuric acid concentration of 1.8% and treatment time of 60 min, the yield of ethanol fermentable sugars (glucose and xylose) was high, (230.5 mg/g dry biomass, comprising 175.2 mg/g glucose and 55.3 mg/g xylose), with 48.6% of total reducing sugars being ethanol fermentable. Therefore, Enteromorpha could be a good candidate for production of fuel ethanol. In future work, the effects of temperature and biomass concentration on hydrolysis, and also the fermentation of the hydrolysates to ethanol fuel should be focused on.

  3. Kinetics of the alkaline hydrolysis of nitrocellulose.

    PubMed

    Christodoulatos, C; Su, T L; Koutsospyros, A

    2001-01-01

    Cellulose nitrate (nitrocellulose) is an explosive solid substance used in large quantities in various formulations of rocket and gun propellants. Safe destruction of nitrocellulose can be achieved by alkaline hydrolysis, which converts it to biodegradable products that can then be treated by conventional biological processes. The kinetics of the alkaline hydrolysis of munitions-grade nitrocellulose in sodium hydroxide solutions were investigated in completely mixed batch reactors. Experiments were conducted using solutions of alkaline strength ranging from 0.1 to 15% by mass and temperatures in the range of 30 to 90 degrees C. Regression analysis of the kinetic data revealed that alkaline hydrolysis of nitrocellulose is of the order 1.0 and 1.5 with respect to nitrocellulose and hydroxide concentration, respectively. The activation energy of the hydrolysis reaction was found to be 100.9 kJ/mol with a preexponential Arrhenius constant of 4.73 x 10(13). Nitrite and nitrate, in a 3:1 ratio, were the primary nitrogen species present in the posthydrolysis solution. The kinetic information is pertinent to the development and optimization of nitrocellulose chemical-biological treatment systems. PMID:11563378

  4. Mechanisms of lactone hydrolysis in acidic conditions.

    PubMed

    Gómez-Bombarelli, Rafael; Calle, Emilio; Casado, Julio

    2013-07-19

    The acid-catalyzed hydrolysis of linear esters and lactones was studied using a hybrid supermolecule-polarizable continuum model (PCM) approach including up to six water molecules. The compounds studied included two linear esters, four β-lactones, two γ-lactones, and one δ-lactone: ethyl acetate, methyl formate, β-propiolactone, β-butyrolactone, β-isovalerolactone, diketene (4-methyleneoxetan-2-one), γ-butyrolactone, 2(5H)-furanone, and δ-valerolactone. The theoretical results are in good quantitative agreement with the experimental measurements reported in the literature and also in excellent qualitative agreement with long-held views regarding the nature of the hydrolysis mechanisms at molecular level. The present results help to understand the balance between the unimolecular (A(AC)1) and bimolecular (A(AC)2) reaction pathways. In contrast to the experimental setting, where one of the two branches is often occluded by the requirement of rather extreme experimental conditions, we have been able to estimate both contributions for all the compounds studied and found that a transition from A(AC)2 to A(AC)1 hydrolysis takes place as acidity increases. A parallel work addresses the neutral and base-catalyzed hydrolysis of lactones. PMID:23731203

  5. Exceptional Amyloid β Peptide Hydrolyzing Activity of Nonphysiological Immunoglobulin Variable Domain Scaffolds*S⃞

    PubMed Central

    Taguchi, Hiroaki; Planque, Stephanie; Sapparapu, Gopal; Boivin, Stephane; Hara, Mariko; Nishiyama, Yasuhiro; Paul, Sudhir

    2008-01-01

    Nucleophilic sites in the paired variable domains of the light and heavy chains (VL and VH domains) of Ig can catalyze peptide bond hydrolysis. Amyloid β (Aβ)-binding Igs are under consideration for immunotherapy of Alzheimer disease. We searched for Aβ-hydrolyzing human IgV domains (IgVs) in a library containing a majority of single chain Fv clones mimicking physiological VL-VH-combining sites and minority IgV populations with nonphysiological structures generated by cloning errors. Random screening and covalent selection of phage-displayed IgVs with an electrophilic Aβ analog identified rare IgVs that hydrolyzed Aβ mainly at His14-Gln15. Inhibition of IgV catalysis and irreversible binding by an electrophilic hapten suggested a nucleophilic catalytic mechanism. Structural analysis indicated that the catalytic IgVs are nonphysiological structures, a two domain heterodimeric VL (IgVL2-t) and single domain VL clones with aberrant polypeptide tags (IgVL-t′). The IgVs hydrolyzed Aβ at rates superior to naturally occurring Igs by 3-4 orders of magnitude. Forced pairing of the single domain VL with VH or VL domains resulted in reduced Aβ hydrolysis, suggesting catalysis by the unpaired VL domain.Ångstrom level amino acid displacements evident in molecular models of the two domain and unpaired VL domain clones explain alterations of catalytic activity. In view of their superior catalytic activity, the VL domain IgVs may help attain clearance of medically important antigens more efficiently than natural Igs. PMID:18974093

  6. Proteins and peptides as renewable flocculants.

    PubMed

    Piazza, G J; Garcia, R A

    2010-08-01

    Partially hydrolyzed extracts from blood meal, feather meal, and meat and bone meal, as well as a variety of common surplus agricultural proteins were tested for their ability to promote the flocculation of clay. Partial alkaline or enzymatic hydrolyses of blood meal, feather meal, and meat and bone meal were performed to liberate proteins and peptides from their water-insoluble forms. Some of these extracts promoted flocculation. However, if hydrolysis was extensive, low molecular weight peptides were mainly produced, and these extracts did not promote flocculation. Beef skin gelatins and hydrolyzed fish collagen were found to promote flocculation when pH 5.5 buffer was added. Commercial preparations of peptone enzymatic digest and a mixture of keratin and hydrolyzed keratin did not promote flocculation. PMID:20236820

  7. Hydrolysis of ketene catalyzed by formic acid: modification of reaction mechanism, energetics, and kinetics with organic acid catalysis.

    PubMed

    Louie, Matthew K; Francisco, Joseph S; Verdicchio, Marco; Klippenstein, Stephen J; Sinha, Amitabha

    2015-05-14

    The hydrolysis of ketene (H2C═C═O) to form acetic acid involving two water molecules and also separately in the presence of one to two water molecules and formic acid (FA) was investigated. Our results show that, while the currently accepted indirect mechanism, involving addition of water across the carbonyl C═O bond of ketene to form an ene-diol followed by tautomerization of the ene-diol to form acetic acid, is the preferred pathway when water alone is present, with formic acid as catalyst, addition of water across the ketene C═C double bond to directly produce acetic acid becomes the kinetically favored pathway for temperatures below 400 K. We find not only that the overall barrier for ketene hydrolysis involving one water molecule and formic acid (H2C2O + H2O + FA) is significantly lower than that involving two water molecules (H2C2O + 2H2O) but also that FA is able to reduce the barrier height for the direct path, involving addition of water across the C═C double bond, so that it is essentially identical with (6.4 kcal/mol) that for the indirect ene-diol formation path involving addition of water across the C═O bond. For the case of ketene hydrolysis involving two water molecules and formic acid (H2C2O + 2H2O + FA), the barrier for the direct addition of water across the C═C double bond is reduced even further and is 2.5 kcal/mol lower relative to the ene-diol path involving addition of water across the C═O bond. In fact, the hydrolysis barrier for the H2C2O + 2H2O + FA reaction through the direct path is sufficiently low (2.5 kcal/mol) for it to be an energetically accessible pathway for acetic acid formation under atmospheric conditions. Given the structural similarity between acetic and formic acid, our results also have potential implications for aqueous-phase chemistry. Thus, in an aqueous environment, even in the absence of formic acid, though the initial mechanism for ketene hydrolysis is expected to involve addition of water across the

  8. GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits

    PubMed Central

    Holtkamp, Wolf; Cunha, Carlos E; Peske, Frank; Konevega, Andrey L; Wintermeyer, Wolfgang; Rodnina, Marina V

    2014-01-01

    Elongation factor G (EF-G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF-G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF-G mutants and translocation-specific antibiotics to investigate timing and energetics of translocation. We show that EF-G–GTP facilitates synchronous movements of peptidyl-tRNA on the two subunits into an early post-translocation state, which resembles a chimeric state identified by structural studies. EF-G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF-G. Our results reveal two distinct modes for utilizing the energy of EF-G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit. PMID:24614227

  9. GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits.

    PubMed

    Holtkamp, Wolf; Cunha, Carlos E; Peske, Frank; Konevega, Andrey L; Wintermeyer, Wolfgang; Rodnina, Marina V

    2014-05-01

    Elongation factor G (EF-G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF-G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF-G mutants and translocation-specific antibiotics to investigate timing and energetics of translocation. We show that EF-G-GTP facilitates synchronous movements of peptidyl-tRNA on the two subunits into an early post-translocation state, which resembles a chimeric state identified by structural studies. EF-G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF-G. Our results reveal two distinct modes for utilizing the energy of EF-G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit. PMID:24614227

  10. Non-catalytic steam hydrolysis of fats

    SciTech Connect

    Deibert, M.C.

    1992-08-28

    Hydrolysis of fats and oils produces fatty acid and glycerol. The catalyzed, liquid phase Colgate-Emry process, state-of-the-art, produces impure products that require extensive energy investment for their purification to commercial grade. Non-catalytic steam hydrolysis may produce products more easily purified. A bench-scale hydrolyzer was designed and constructed to contact descending liquid fat or oil with rising superheated steam. Each of the five stages in the reactor was designed similar to a distillation column stage to promote intimate liquid-gas contact. Degree of hydrolysis achieved in continuous tests using tallow feed were 15% at 280C and 35% at 300C at a tallow-to-steam mass feed ratio of 4.2. At a feed ratio of 9.2, the degree of hydrolysis was 21% at 300C. Decomposition was strongly evident at 325C but not at lower temperatures. Soybean oil rapidly polymerized under reaction conditions. Batch tests at 320C produced degrees of hydrolyses of between 44% and 63% using tallow and palm oil feeds. Over 95% fatty acids were present in a clean, readily separated organic portion of the overhead product from most tests. The test reactor had serious hydraulic resistance to liquid down-flow which limited operation to very long liquid residence times. These times are in excess of those that tallow and palm oil are stable at the reaction temperature. Little glycerol and extensive light organics were produced indicating that unexplained competing reactions to hydrolysis occurred in the experimental system. Further tests using an improved reactor will be required.

  11. Novel peptide recognized by RhoA GTPase.

    PubMed

    Drulis-Fajdasz, Dominika; Jelen, Filip; Oleksy, Arkadiusz; Otlewski, Jacek

    2006-01-01

    A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA-R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+-and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein. PMID:17019437

  12. Theoretical aspects of the biological catch bond.

    PubMed

    Prezhdo, Oleg V; Pereverzev, Yuriy V

    2009-06-16

    bond properties on weak protein-water interactions may provide universal activation mechanisms in many biological systems and create new types of catch binding. Molecular dynamics simulations provide atomistic insights: the molecular view of bond dissociation gives a foundation for theoretical models and differentiates between alternative interpretations of experimental data. The number of known catch bonds is growing; analogs are found in enzyme catalysis, peptide translocation through nanopores, DNA unwinding, photoinduced dissociation of chemical bonds, and negative thermal expansion of bulk materials, for example. Finer force resolution will likely provide many more. Understanding the properties of catch bonds offers insight into the behavior of biological systems subjected to external perturbations in general. PMID:19331389

  13. Synthesis and evaluation of amphiphilic peptides as nanostructures and drug delivery tools

    NASA Astrophysics Data System (ADS)

    Sayeh, Naser Ali

    conjugates although one limitation lies in the effort of controlling the rate of drug release. The encapsulated or complexed drugs tend to be released rapidly (before reaching the target site) and in the dendrimer--drug conjugates, it is the chemical linkage that controls the drug release. Thus, future studies in this field are urgently required to create more efficient and stable biomaterials. Peptides are considered as efficient vectors for achieving optimal cellular uptake. The potential use of peptides as drug delivery vectors received much attention by the discovery of several cell-penetrating peptides (CPPs). The first CPPs discovered in 1988, that were sequences from HIV-1 encoded TAT protein, TAT (48--60), and penetrated very efficiently through cell membranes of cultured mammalian cells. CPPs are a class of diverse peptides, typically with 8--25 amino acids, and unlike most peptides, they can cross the cellular membrane with more efficiency. CPPs have also shown to undergo self-assembly and generate nanostructures. The generation of self-assembled peptides and nanostructures occur through various types of interactions between functional groups of amino acid residues, such as electrostatic, hydrophobic, and hydrogen bonding. Appropriate design and functionalization of peptides are critical for generating nanostructures. Chemically CPPs are classified into two major groups: linear and cyclic peptides. It has been previously reported that linear peptides containing hydrophilic and hydrophobic amino acids could act as membrane protein stabilizers. These compounds are short hydrophilic or amphiphilic peptides that have positively charged amino acids, such as arginine, lysine or histidine, which can interact with the negative charge phospholipids layer on the cell membrane and translocate the cargo into the cells. Conjugation to cationic linear CPPs, such as TAT, penetratin, or oligoarginine efficiently improves the cellular uptake of large hydrophilic molecules, but the

  14. Possible involvement of the A/sup 20/-A/sup 21/ peptide bond in the expression of the biological activity of insulin. 1. (21-Desasparagine,20-cysteinamide-A)insulin and (21-desasparagine,20-cysteine isopropylamide-A)insulin

    SciTech Connect

    Chu, Y.C.; Wang, R.Y.; Burke, G.T.; Chanley, J.D.; Katsoyannis, P.G.

    1987-11-03

    The C-terminal region of the A chain of insulin has been shown to play a significant role in the expression of the biological activity of the hormone. To further delineate the contribution of this segment, we have synthesized (21-desasparagine,20-cysteinamide-A)insulin and (21-desasparagine,20-cysteine isopropylamide-A)insulin, in which the C-terminal amino acid residue of the A chain of insulin, asparagine, has been removed and the resulting free carboxyl group of the A/sup 20/ cysteine residue has been converted to an amide and an isopropylamide, respectively. Both insulin analogues display biological activity, 14-15% for the unsubstituted amide analogue and 20-22% for the isopropylamide analogue, both relative to bovine insulin. In contrast, a (21-desasparagine-A)insulin analogue has been reported to display less than 4% of the activity of the natural hormone. The implications of these findings are discussed, and we conclude that the A/sup 20/-A/sup 21/ amide bond plays a significant role in the expression of the biological activity of insulin.

  15. QSAR for cholinesterase inhibition by organophosphorus esters and CNDO/2 calculations for organophosphorus ester hydrolysis

    NASA Technical Reports Server (NTRS)

    Johnson, H.; Kenley, R. A.; Rynard, C.; Golub, M. A.

    1985-01-01

    Quantitative structure-activity relationships were derived for acetyl- and butyrylcholinesterase inhibition by various organophosphorus esters. Bimolecular inhibition rate constants correlate well with hydrophobic substituent constants, and with the presence or absence of catonic groups on the inhibitor, but not with steric substituent constants. CNDO/2 calculations were performed on a separate set of organophosphorus esters, RR'P(O)X, where R and R' are alkyl and/or alkoxy groups and X is fluorine, chlorine or a phenoxy group. For each subset with the same X, the CNDO-derived net atomic charge at the central phosphorus atom in the ester correlates well with the alkaline hydrolysis rate constant. For the whole set of esters with different X, two equations were derived that relate either charge and leaving group steric bulk, or orbital energy and bond order to the hydrogen hydrolysis rate constant.

  16. Nanocellulose prepared by acid hydrolysis of isolated cellulose from sugarcane bagasse

    NASA Astrophysics Data System (ADS)

    Wulandari, W. T.; Rochliadi, A.; Arcana, I. M.

    2016-02-01

    Cellulose in nanometer range or called by nano-cellulose has attracted much attention from researchers because of its unique properties. Nanocellulose can be obtained by acid hydrolysis of cellulose. The cellulose used in this study was isolated from sugarcane bagasse, and then it was hydrolyzed by 50% sulfuric acid at 40 °C for 10 minutes. Nanocellulose has been characterized by Transmission Electron Microscope (TEM), Particle Size Analyzer (PSA), Fourier Transform Infrared Spectroscopy (FTIR) and X-Ray Diffraction (XRD). Analysis of FTIR showed that there were not a new bond which formed during the hydrolysis process. Based on the TEM analysis, nano-cellulose has a spherical morphology with an average diameter of 111 nm and a maximum distribution of 95.9 nm determined by PSA. The XRD analysis showed that the crystallinity degree of nano-cellulose was higher than cellulose in the amount of 76.01%.

  17. Optimization of synergism of a recombinant auxiliary activity 9 from Chaetomium globosum with cellulase in cellulose hydrolysis.

    PubMed

    Kim, In Jung; Nam, Ki Hyun; Yun, Eun Ju; Kim, Sooah; Youn, Hak Jin; Lee, Hee Jin; Choi, In-Geol; Kim, Kyoung Heon

    2015-10-01

    Auxiliary activity family 9 (AA9, formerly known as glycoside hydrolase family 61 or polysaccharide monooxygenase) is a group of fungal proteins that were recently found to have a significant synergism with cellulase in cellulose hydrolysis via the oxidative cleavage of glycosidic bonds of cellulose chains. In this study, we report the active expression of a recombinant fungal AA9 from Chaetomium globosum (CgAA9) in a bacterial host, Escherichia coli, and the optimization of its synergistic activity in cellulose hydrolysis by using cellulase. The recombinant CgAA9 (0.9 mg/g cellulose) exhibited 1.7-fold synergism in the hydrolysis of Avicel when incubated with 0.9 filter paper units of Celluclast 1.5 L/g cellulose. The first study of the active expression of AA9 using a bacterial host and its synergistic optimization could be useful for the industrial application of AA9 for the saccharification of lignocellulose. PMID:25936375

  18. Small peptides as potent mimetics of the protein hormone erythropoietin.

    PubMed

    Wrighton, N C; Farrell, F X; Chang, R; Kashyap, A K; Barbone, F P; Mulcahy, L S; Johnson, D L; Barrett, R W; Jolliffe, L K; Dower, W J

    1996-07-26

    Random phage display peptide libraries and affinity selective methods were used to isolate small peptides that bind to and activate the receptor for the cytokine erythropoietin (EPO). In a panel of in vitro biological assays, the peptides act as full agonists and they can also stimulate erythropoiesis in mice. These agonists are represented by a 14- amino acid disulfide-bonded, cyclic peptide with the minimum consensus sequence YXCXXGPXTWXCXP, where X represents positions allowing occupation by several amino acids. The amino acid sequences of these peptides are not found in the primary sequence of EPO. The signaling pathways activated by these peptides appear to be identical to those induced by the natural ligand. This discovery may form the basis for the design of small molecule mimetics of EPO. PMID:8662529

  19. Technical bases for precipitate hydrolysis process operating parameters

    SciTech Connect

    Bannochie, C.J.

    1992-10-05

    This report provides the experimental data and rationale in support of the operating parameters for precipitate hydrolysis specified in WSRC-RP-92737. The report is divided into two sections, the first dealing with lab-scale precipitate hydrolysis experimentation while the second part addresses large-scale runs conducted to demonstrate the revised operating parameters in the Precipitate Hydrolysis Experimental Facility (PHEF).

  20. Molecular Investigation of the Mechanism of Non-Enzymatic Hydrolysis of Proteins and the Predictive Algorithm for Susceptibility.

    PubMed

    Lauer, Timothy M; Wood, Geoffrey P F; Farkas, David; Sathish, Hasige A; Samra, Hardeep S; Trout, Bernhardt L

    2016-06-14

    A number of potential degradation routes can limit the shelf life of a biotherapeutic. While these are experimentally measurable, the tests to do so require a substantial investment in both time and material, resources rarely available early in the drug development process. To address the potential degradation route of non-enzymatic hydrolysis, we performed a molecular modeling analysis, together with an experimental study, to gain detailed insight into the reaction. On the basis of the mechanism, an algorithm for predicting the likely cleavage sites of a protein has been created. This algorithm measures four key properties during a molecular dynamics simulation, which relate to the key steps of the hydrolysis mechanism, in particular the rate-determining step (which can vary depending on the local environment). The first two properties include the secondary structure and the surface exposure of the amide bond, both of which help detect if the addition of the proton to the amide bond is possible. The second two properties relate to whether the side chain can cyclize and form a furane ring. These two properties are the orientation of the side chain relative to the amide bond and the number of hydrogen bonds between the side chain and the surrounding protein. Overall, the algorithm performs well at identifying reactive versus nonreactive bonds. The algorithm correctly classifies nearly 90% of all amide bonds following an aspartic or glutamic acid residue as reactive or nonreactive. PMID:27194363

  1. Chemical Bonds II

    ERIC Educational Resources Information Center

    Sanderson, R. T.

    1972-01-01

    The continuation of a paper discussing chemical bonding from a bond energy viewpoint, with a number of examples of single and multiple bonds. (Part I appeared in volume 1 number 3, pages 16-23, February 1972.) (AL)

  2. What Determines Bond Costs. Municipal Bonds Series.

    ERIC Educational Resources Information Center

    Young, Douglas; And Others

    Public officials in small towns who participate infrequently in the bond market need information about bond financing. This publication, one in a series of booklets published by the Western Rural Development Center using research gathered between 1967-77, discusses factors influencing the marketability and cost of bond financing for towns and…

  3. Orchestration of Structural, Stereoelectronic, and Hydrogen-Bonding Effects in Stabilizing Triplexes from Engineered Chimeric Collagen Peptides (Pro(X)-Pro(Y)-Gly)6 Incorporating 4(R/S)-Aminoproline.

    PubMed

    Umashankara, Muddegowda; Sonar, Mahesh V; Bansode, Nitin D; Ganesh, Krishna N

    2015-09-01

    Collagens are an important family of structural proteins found in the extracellular matrix with triple helix as the characteristic structural motif. The collagen triplex is made of three left-handed polyproline II (PPII) helices with each PPII strand consisting of repetitive units of the tripeptide motif X-Y-Gly, where the amino acids X and Y are most commonly proline (Pro) and 4R-hydroxyproline (Hyp), respectively. A C4-endo pucker at X-site and C4-exo pucker at Y-site have been proposed to be the key for formation of triplex, and the nature of pucker is dependent on both the electronegativity and stereochemistry of the substituent. The present manuscript describes a new class of collagen analogues-chimeric cationic collagens-wherein both X- and Y-sites in collagen triad are simultaneously substituted by a combination of 4(R/S)-(OH/NH2/NH3(+)/NHCHO)-prolyl units and triplex stabilities measured at different pHs and in EG:H2O. Based on the results a model has been proposed with the premise that any factors which specifically favor the ring puckers of C4-endo at X-site and C4-exo at Y-site stabilize the PPII conformation and hence the derived triplexes. The pH-dependent triplex stability uniquely observed with ionizable 4-amino substituent on proline enables one to define the critical combination of factors C4-(exo/endo), intraresidue H-bonding, stereoelectronic (R/S) and n → π* interactions in dictating the triplex strength. The ionizable NH2 substituent at C4 in R/S configuration is thus a versatile probe for delineating the triplex stabilizing factors and the results have potential for designing of collagen analogues with customized properties for material and biological applications. PMID:26274096

  4. Production of bioactive peptide hydrolysates from deer, sheep and pig plasma using plant and fungal protease preparations.

    PubMed

    Bah, Clara S F; Bekhit, Alaa El-Din A; Carne, Alan; McConnell, Michelle A

    2015-06-01

    Plasma separated from deer, sheep and pig blood, obtained from abattoirs, was hydrolysed using protease preparations from plant (papain and bromelain) and fungal (FP400 and FPII) sources. Antioxidant and antimicrobial activities of the peptide hydrolysates obtained after 1, 2, 4 and 24h of hydrolysis, were investigated. The release of trichloroacetic acid-soluble peptides over the hydrolysis period was monitored using the o-phthaldialdehyde (OPA) assay, while the hydrolysis profiles were visualised using SDS-PAGE. The major plasma proteins in the animal plasmas were identified using MALDI-TOF-TOF MS. Hydrolysates of plasma generated with fungal proteases exhibited higher DPPH radical-scavenging, oxygen radical-scavenging capacity (ORAC) and ferric reducing antioxidant power (FRAP) than those generated with plant proteases for all three animal plasmas. No antimicrobial activity was detected in the hydrolysates. The results indicated that proteolytic hydrolysis of animal blood plasmas, using fungal protease preparations in particular, produces hydrolysates with high antioxidant properties. PMID:25624206

  5. Higher efficiency soluble prokaryotic expression, purification, and structural analysis of antimicrobial peptide G13.

    PubMed

    Che, Yuanyuan; Lu, Yinghu; Zha, Xiangdong; Huang, Huoqing; Yang, Peilong; Ma, Lijuan; Xu, Xuejiao

    2016-03-01

    G13 is a 19-residue cationic antimicrobial peptide derived from granulysin. In order to achieve high-level expression of G13 in Escherichia coli cells, and to reduce downstream processing costs, we introduced an Asp-Pro acid labile bond between the His-Patch thioredoxin and G13 and constructed the recombinant plasmid pThiohisA-DP-G13. The plasmid was transformed into E. coli BL21 (DE3). After induction with isopropyl-β-d-thiogalactopyranoside for 5 h, the fusion protein accumulated up to 200 mg/L in soluble form. The fusion protein was released by a high pressure homogenizer, cleaved using 13% acetic acid at 50 °C hydrolysis for 72 h. The recombinant G13 (r-G13) was then successively purified by fractional precipitation with ammonium sulfate and trichloroacetic acid, followed by one-step cation exchange chromatography. The purified r-G13 displayed a single band (about 2.2 kDa) as analyzed by Tris-Tricine buffered SDS-PAGE, and its precise molecular weight was confirmed using tandem mass spectrometry. Analysis of r-G13 by circular dichroism (CD) indicated that r-G13 contained predominantly β-sheet and random coil. Agar plate diffusion assay revealed that the r-G13 exhibited antibacterial activity against both Bacillus subtilis and E. coli. PMID:26581777

  6. Mass Spectrometric Strategies to Improve the Identification of Pt(II)-Modification Sites on Peptides and Proteins

    NASA Astrophysics Data System (ADS)

    Li, Huilin; Snelling, Jonathon R.; Barrow, Mark P.; Scrivens, James H.; Sadler, Peter J.; O'Connor, Peter B.

    2014-07-01

    To further explore the binding chemistry of cisplatin ( cis-Pt(NH3)2Cl2) to peptides and also establish mass spectrometry (MS) strategies to quickly assign the platinum-binding sites, a series of peptides with potential cisplatin binding sites (Met(S), His(N), Cys(S), disulfide, carboxyl groups of Asp and Glu, and amine groups of Arg and Lys, were reacted with cisplatin, then analyzed by electron capture dissociation (ECD) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). Radical-mediated side-chain losses from the charge-reduced Pt-binding species (such as CH3S• or CH3SH from Met, SH• from Cys, CO2 from Glu or Asp, and NH2 • from amine groups) were found to be characteristic indicators for rapid and unambiguous localization of the Pt-binding sites to certain amino acid residues. The method was then successfully applied to interpret the top-down ECD spectrum of an inter-chain Pt-crosslinked insulin dimer, insulin + Pt(NH3)2 + insulin (>10 kDa). In addition, ion mobility MS shows that Pt binds to multiple sites in Substance P, generating multiple conformers, which can be partially localized by collisionally activated dissociation (CAD). Platinum(II) (Pt(II)) was found to coordinate to amine groups of Arg and Lys, but not to disulfide bonds under the conditions used. The coordination of Pt to Arg or Lys appears to arise from the migration of Pt(II) from Met(S) as shown by monitoring the reaction products at different pH values by ECD. No direct binding of cisplatin to amine groups was observed at pH 3 ~ 10 unless Met residues were present in the sequence, but noncovalent interactions between cisplatin hydrolysis and amination [Pt(NH3)4]2+ products and these peptides were found regardless of pH.

  7. Aluminum Nitride Hydrolysis Enabled by Hydroxyl-Mediated Surface Proton Hopping.

    PubMed

    Bartel, Christopher J; Muhich, Christopher L; Weimer, Alan W; Musgrave, Charles B

    2016-07-20

    Aluminum nitride (AlN) is used extensively in the semiconductor industry as a high-thermal-conductivity insulator, but its manufacture is encumbered by a tendency to degrade in the presence of water. The propensity for AlN to hydrolyze has led to its consideration as a redox material for solar thermochemical ammonia (NH3) synthesis applications where AlN would be intentionally hydrolyzed to produce NH3 and aluminum oxide (Al2O3), which could be subsequently reduced in nitrogen (N2) to reform AlN and reinitiate the NH3 synthesis cycle. No quantitative, atomistic mechanism by which AlN, and more generally, metal nitrides react with water to become oxidized and generate NH3 yet exists. In this work, we used density-functional theory (DFT) to examine the reaction mechanisms of the initial stages of AlN hydrolysis, which include: water adsorption, hydroxyl-mediated proton diffusion to form NH3, and NH3 desorption. We found activation barriers (Ea) for hydrolysis of 330 and 359 kJ/mol for the cases of minimal adsorbed water and additional adsorbed water, respectively, corroborating the high observed temperatures for the onset of steam AlN hydrolysis. We predict AlN hydrolysis to be kinetically limited by the dissociation of strong Al-N bonds required to accumulate protons on surface N atoms to form NH3. The hydrolysis mechanism we elucidate is enabled by the diffusion of protons across the AlN surface by a hydroxyl-mediated Grotthuss mechanism. A comparison between intrinsic (Ea = 331 kJ/mol) and mediated proton diffusion (Ea = 89 kJ/mol) shows that hydroxyl-mediated proton diffusion is the predominant mechanism in AlN hydrolysis. The large activation barrier for NH3 generation from AlN (Ea = 330 or 359 kJ/mol, depending on water coverage) suggests that in the design of materials for solar thermochemical ammonia synthesis, emphasis should be placed on metal nitrides with less covalent metal-nitrogen bonds and, thus, more-facile NH3 liberation. PMID:27341277

  8. Fatty acid specificity of hormone-sensitive lipase. Implication in the selective hydrolysis of triacylglycerols.

    PubMed

    Raclot, T; Holm, C; Langin, D

    2001-12-01

    The selective mobilization of fatty acids from white fat cells depends on their molecular structure, in particular the degree of unsaturation. The present study was designed to examine if the release of fatty acids by hormone-sensitive lipase (HSL) in vitro i) is influenced by the amount of unsaturation, ii) depends on the temperature, and iii) could explain the selective pattern of fatty acid mobilization and notably the preferential mobilization of certain highly unsaturated fatty acids. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 35 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation from 0 to 6 double bonds was measured. Fatty acid composition of in vitro released NEFA was compared with that of fat cell triacylglycerols (TAG), the ratio % NEFA/% TAG being defined as the relative hydrolysis. The relative hydrolysis of individual fatty acids differed widely, ranging from 0.44 (24:1n-9) to 1.49 (18:1n-7) with rat HSL, and from 0.38 (24:1n-9) to 1.67 (18:1n-7) with human HSL. No major difference was observed between rat and human HSL. The relative release was dependent on the number of double bonds according to chain length. The amount of fatty acid released by recombinant rat HSL was decreased but remained robust at 4 degrees C compared with 37 degrees C, and the relative hydrolysis of some individual fatty acids was affected. The relative hydrolysis of fatty acids moderately, weakly, and highly mobilized by adipose tissue in vivo was similar and close to unity in vitro. We conclude that i) the release of fatty acids by HSL is only slightly affected by their degree of unsaturation, ii) the ability of HSL to efficiently and selectively release fatty acids at low temperature could reflect a cold adaptability for poikilotherms or hibernators when endogenous lipids are needed, and iii) the selectivity of fatty acid hydrolysis by HSL does not fully account for the selective pattern of

  9. Halogen bonds in biological molecules

    PubMed Central

    Auffinger, Pascal; Hays, Franklin A.; Westhof, Eric; Ho, P. Shing

    2004-01-01

    Short oxygen–halogen interactions have been known in organic chemistry since the 1950s and recently have been exploited in the design of supramolecular assemblies. The present survey of protein and nucleic acid structures reveals similar halogen bonds as potentially stabilizing inter- and intramolecular interactions that can affect ligand binding and molecular folding. A halogen bond in biomolecules can be defined as a short CX···OY interaction (CX is a carbon-bonded chlorine, bromine, or iodine, and OY is a carbonyl, hydroxyl, charged carboxylate, or phosphate group), where the X···O distance is less than or equal to the sums of the respective van der Waals radii (3.27 Å for Cl···O, 3.37Å for Br···O, and 3.50 Å for I···O) and can conform to the geometry seen in small molecules, with the CX···O angle ≈165° (consistent with a strong directional polarization of the halogen) and the X···OY angle ≈120°. Alternative geometries can be imposed by the more complex environment found in biomolecules, depending on which of the two types of donor systems are involved in the interaction: (i) the lone pair electrons of oxygen (and, to a lesser extent, nitrogen and sulfur) atoms or (ii) the delocalized π -electrons of peptide bonds or carboxylate or amide groups. Thus, the specific geometry and diversity of the interacting partners of halogen bonds offer new and versatile tools for the design of ligands as drugs and materials in nanotechnology. PMID:15557000

  10. Halogen bonds in biological molecules.

    PubMed

    Auffinger, Pascal; Hays, Franklin A; Westhof, Eric; Ho, P Shing

    2004-11-30

    Short oxygen-halogen interactions have been known in organic chemistry since the 1950s and recently have been exploited in the design of supramolecular assemblies. The present survey of protein and nucleic acid structures reveals similar halogen bonds as potentially stabilizing inter- and intramolecular interactions that can affect ligand binding and molecular folding. A halogen bond in biomolecules can be defined as a short C-X...O-Y interaction (C-X is a carbon-bonded chlorine, bromine, or iodine, and O-Y is a carbonyl, hydroxyl, charged carboxylate, or phosphate group), where the X...O distance is less than or equal to the sums of the respective van der Waals radii (3.27 A for Cl...O, 3.37 A for Br...O, and 3.50 A for I...O) and can conform to the geometry seen in small molecules, with the C-X...O angle approximately 165 degrees (consistent with a strong directional polarization of the halogen) and the X...O-Y angle approximately 120 degrees . Alternative geometries can be imposed by the more complex environment found in biomolecules, depending on which of the two types of donor systems are involved in the interaction: (i) the lone pair electrons of oxygen (and, to a lesser extent, nitrogen and sulfur) atoms or (ii) the delocalized pi -electrons of peptide bonds or carboxylate or amide groups. Thus, the specific geometry and diversity of the interacting partners of halogen bonds offer new and versatile tools for the design of ligands as drugs and materials in nanotechnology. PMID:15557000

  11. Cationic gemini surfactants with cleavable spacer: chemical hydrolysis, biodegradation, and toxicity.

    PubMed

    Tehrani-Bagha, A R; Holmberg, K; van Ginkel, C G; Kean, M

    2015-07-01

    The paper describes synthesis and characterization of a new type of cationic gemini surfactant, which has dodecyl tails and a spacer that contains an ester bond. The nomenclature used to describe the structure is 12Q2OCO1Q12, with Q being a quaternary ammonium group and the numbers indicating the number of methylene or methyl groups. Due to the close proximity to the two quaternary ammonium groups, the ester bond is very stable on the acid side and very labile already at slightly alkaline conditions. The hydrolysis products are two single chain surfactants (i.e. 12Q2OH and 12Q1COOH) which are less surface active than the intact gemini surfactant. 12Q2OCO1Q12 was found to be readily biodegradable, i.e. it gave more than 60% biodegradation after 28 days. This is interesting because similar gemini surfactants but with ester bonds in the tails instead of the spacer, have previously been found not to be readily biodegradable. The gemini surfactant was found to be toxic to aquatic organisms (ErC50 value of 0.27 mg/l), although less toxic than the two hydrolysis products. PMID:25446957

  12. Continuous steam hydrolysis of tulip poplar

    SciTech Connect

    Fieber, C.; Colcord, A.R.; Faass, S.; Muzzy, J.D.; Roberts, R.S.

    1982-08-01

    To produce ethanol from hardwood it is desirable to fractionate the hardwood in order to produce a relatively pure cellulosic pulp for dilute acid hydrolysis. An experimental investigation of continuous steam hydrolysis of tulip poplar wood chips indicates that over 90% of the lignin present can be extracted by 0.1N sodium hydroxide, resulting in a cellulose pulp containing over 90% hexosan. The study was performed using a Stake Technology, Ltd., continuous digester rated at one oven dry ton per hour of wood chips. The yields of hexosans, hexoses, xylan, xylose, lignin, furfural, acetic acid and methanol were determined as a function of residence time and steam pressure in the digester. The information provides a basis for establishing a material and energy balance for a hardwood to ethanol plant.

  13. Pretreatment of sallow prior to enzymatic hydrolysis

    SciTech Connect

    Galbe, M.; Zacchi, G.; Scott, C.D.

    1986-01-01

    Pretreatment of fast-growing sallow by steam explosion prior to enzymic hydrolysis was investigated to find optimum conditions regarding pretreatment temperature and time. Some preliminary experiments with impregnation of the material with H/sub 2/SO/sub 4/ or Na/sub 2/SO/sub 3/ were performed to reduce the byproduct formation and to increase the xylose yield. A temperature of 220 degrees for 15 minutes gave the highest yield, approximately 80% of the glucose available based on raw material. The xylose recovered was equal to or less than 20% when no chemicals were added. Impregnation with Na/sub 2/SO/sub 3/ gave an improvement compared with the unimpregnated material. About 30% of the xylose content could thus be recovered after the enzymic hydrolysis. The results are promising. (Refs. 5).

  14. Pathway of processive ATP hydrolysis by kinesin

    PubMed Central

    Gilbert, Susan P.; Webb, Martin R.; Brune, Martin; Johnson, Kenneth A.

    2007-01-01

    Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle. The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration. Kinesin dissociates from the microtubule after ATP hydrolysis. This step is rate-limiting. The subsequent rebinding of kinesin · ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state. These results provide an explanation for the motility differences between skeletal myosin and kinesin. PMID:7854446

  15. Hydrolysis of the RNA model substrate catalyzed by a binuclear Zr(IV)-substituted Keggin polyoxometalate.

    PubMed

    Luong, Thi Kim Nga; Absillis, Gregory; Shestakova, Pavletta; Parac-Vogt, Tatjana N

    2015-09-21

    The reactivity and solution behaviour of the binuclear Zr(IV)-substituted Keggin polyoxometalate (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]·7H2O (ZrK 2 : 2) towards phosphoester bond hydrolysis of the RNA model substrate 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP) was investigated at different reaction conditions (pD, temperature, concentration, and ionic strength). The hydrolysis of the phosphoester bond of HPNP, followed by means of (1)H NMR spectroscopy, proceeded with an observed rate constant, kobs = 11.5(±0.42) × 10(-5) s(-1) at pD 6.4 and 50 °C, representing a 530-fold rate enhancement in comparison with the spontaneous hydrolysis of HPNP. (1)H and (31)P NMR spectra indicate that at these reaction conditions the only products of hydrolysis are p-nitrophenol and the corresponding cyclic phosphate ester. The pD dependence of kobs exhibits a bell-shaped profile, with the fastest rate observed at pD 6.4. The formation constant (Kf = 455 M(-1)) and catalytic rate constant (kc = 42 × 10(-5) s(-1)) for the HPNP-ZrK 2 : 2 complex, activation energy (Ea) of 63.35 ± 1.82 kJ mol(-1), enthalpy of activation (ΔH(‡)) of 60.60 ± 2.09 kJ mol(-1), entropy of activation (ΔS(‡)) of -133.70 ± 6.13 J mol(-1) K(-1), and Gibbs activation energy (ΔG(‡)) of 102.05 ± 0.13 kJ mol(-1) at 37 °C were calculated from kinetic experiments. Binding between ZrK 2 : 2 and the P-O bond of HPNP was evidenced by the change in the (31)P chemical shift and signal line-broadening of the (31)P atom in HPNP upon addition of ZrK 2 : 2. Based on (31)P NMR experiments and isotope effect studies, a mechanism for HPNP hydrolysis in the presence of ZrK 2 : 2 was proposed. PMID:26256057

  16. PLA recycling by hydrolysis at high temperature

    NASA Astrophysics Data System (ADS)

    Cristina, Annesini Maria; Rosaria, Augelletti; Sara, Frattari; Fausto, Gironi

    2016-05-01

    In this work the process of PLA hydrolysis at high temperature was studied, in order to evaluate the possibility of chemical recycling of this polymer bio-based. In particular, the possibility to obtain the monomer of lactic acid from PLA degradation was investigated. The results of some preliminary tests, performed in a laboratory batch reactor at high temperature, are presented: the experimental results show that the complete degradation of PLA can be obtained in relatively low reaction times.

  17. A dash of protons: A theoretical study on the hydrolysis mechanism of 1-substitued silatranes and their protonated analogs

    SciTech Connect

    Sok, Sarom; Gordon, Mark

    2011-08-17

    Ab initio calculations were carried out to study the hydrolysis mechanism of 1-substituted silatranes in the presence of an acid (acid-catalyzed) and an additional water (water-assisted). Compared with the neutral hydrolysis mechanism involving one water, use of an acid catalyst reduces the barrier associated with the rate-limiting step by approximate to 14 kcal/mol. A modest decrease of approximate to 5 kcal/mol is predicted when an additional water molecule is added to the neutral hydrolysis mechanism involving one water. The combination of an acid catalyst and an additional water molecule reduces the barrier by approximate to 27 kcal/mol. Bond order analysis suggests ring cleavage involving the bond breaking of a siloxane and silanol group during the neutral and acid-catalyzed hydrolysis of 1-substituted silatranes. respectively. Solvent effects, represented by the PCM continuum model, do not qualitatively alter computational gas-phase results. (C) 2011 Elsevier B.V. All rights reserved.

  18. Studies on the carbohydrate moiety of α1-acid glycoprotein (orosomucoid) by using alkaline hydrolysis and deamination by nitrous acid

    PubMed Central

    Isemura, M.; Schmid, K.

    1971-01-01

    Alkaline hydrolysis followed by deamination with nitrous acid was applied for the first time to a glycoprotein, human plasma α1-acid glycoprotein (orosomucoid). This procedure, which specifically cleaves the glycosaminidic bonds, yielded well-defined oligosaccharides. The trisaccharides, which were obtained from the native protein, consisted of a sialic acid derivative, galactose and 2,5-anhydromannose. The linkage between galactose and 2,5-anhydromannose is most probably a (1→4)-glycosidic bond. A hitherto unknown linkage between N-acetylneuraminic acid and galactose was also established, namely a (2→2)-linkage. The three linkages between sialic acid and galactose described in this paper appear to be about equally resistant to mild acid hydrolysis. The disaccharide that was derived from the desialized glycoprotein consisted of galactose and 2,5-anhydromannose. Evidence was obtained for the presence of a new terminal sialyl→N-acetylglucosamine disaccharide accounting for approximately 1mol/mol of protein. The presence of this disaccharide may explain the relatively severe requirements for the complete acid hydrolysis of the sialyl residues. The present study indicates that alkaline hydrolysis followed by nitrous acid deamination in conjunction with gas–liquid chromatography will afford relatively rapid determination of the partial structure of the complex carbohydrate moiety of glycoproteins. PMID:5135244

  19. Hydrolysis of wheat gluten by combining peptidases of Flammulina velutipes and electrodialysis.

    PubMed

    Giesler, Lucienne; Linke, Diana; Rabe, Swen; Appel, Daniel; Berger, Ralf Günter

    2013-09-11

    Wheat gluten hydrolysis, used to generate seasonings, was studied using peptidases from Flammulina velutipes or commercial Flavourzyme. L-amino acids were added in a range from 0.5 to 75.0 mM, and L-isoleucine, L-leucine, L-valine, and L-phenylalanine were identified as the strongest inhibitors for both enzyme mixtures. L-serine inhibited Flammulina velutipes peptidases only, while L-histidine and L-glutamine inhibited Flavourzyme peptidases only. To reduce product inhibition by released L-amino acids, electrodialysis was explored. An increase of the degree of hydrolysis of up to 60% for Flammulina velutipes peptidases and 31% for Flavourzyme compared to that for the best control batch was observed after applying an electrodialysis unit equipped with an ultrafiltration membrane for two times 1 h during the 20 h of hydrolysis. The total transfer of free L-amino acids into the concentrate reached 25-30% per hour. Peptides passed the membrane less easily, although the nominal cutoff was 4 kDa. PMID:23947566

  20. Enzymatic hydrolysis of starry triggerfish (Abalistes stellaris) muscle using liver proteinase from albacore tuna (Thunnus alalunga).

    PubMed

    Sripokar, P; Chaijan, M; Benjakul, S; Kishimura, H; Klomklao, S

    2016-02-01

    Proteinases from liver extract from albacore tuna (Thunnus alalunga) were used to produce protein hydrolysate from starry triggerfish (Abalistes stellaris) muscle. Hydrolysis conditions for preparing protein hydrolysate from starry triggerfish muscle were optimized. Enzyme level, reaction time and fish muscle/buffer ratio significantly affected the hydrolysis (p < 0.05). Optimum conditions for triggerfish muscle hydrolysis were 5.5 % liver extract, 40 min reaction time and fish muscle/buffer ratio of 1:3 (w/v). The freeze-dried protein hydrolysate was characterized with respect to chemical composition, amino acid composition and color. The product contained 91.73 % protein, 2.04 % lipid and 6.48 % ash. The protein hydrolysate exhibited high amount of essential amino acids (45.62 %). It was light yellow in color (L (*) = 82.94, a (*) = 0.84, b (*) = 22.83). The results indicate that the extract from liver of albacore tuna could be used to produce fish protein hydrolysate and protein hydrolysate from starry triggerfish muscle may potentially serve as a good source of desirable peptide and amino acids. PMID:27162384

  1. Coupling of ultrafiltration and enzymatic hydrolysis aiming at valorizing shrimp wastewater.

    PubMed

    Tonon, Renata V; dos Santos, Bianca A; Couto, Cinthia C; Mellinger-Silva, Caroline; Brígida, Ana Iraidy S; Cabral, Lourdes M C

    2016-05-01

    The objective of this work was to obtain a protein hydrolysate from the wastewater generated during shrimp cooking, by coupling ultrafiltration and enzymatic hydrolysis processes. Initially, the effluent was concentrated by ultrafiltration, reaching a protein concentration factor of 3.2. The concentrated effluent was then enzymatically hydrolyzed, aiming at obtaining peptides with antioxidant capacity. The effects of some process variables--temperature (55-75 °C), pH (7-9) and enzyme/substrate (E/S) ratio (0.1-2.5%)--on the degree of hydrolysis and the antioxidant capacity were evaluated. The increase in temperature and pH resulted in lower degree of hydrolysis and higher antioxidant capacity. The conditions selected as the most suitable were: temperature of 75 °C, pH of 9.0 and E/S ratio of 0.1%. The hydrolysates produced at these conditions were also evaluated for total amino acid content and electrophoretic profile, showing a suitable amount of essential amino acids that covers the recommended daily needs. PMID:26769500

  2. Role of ATP binding and hydrolysis in assembly of MacAB-TolC macrolide transporter.

    PubMed

    Lu, Shuo; Zgurskaya, Helen I

    2012-12-01

    MacB is a founding member of the Macrolide Exporter family of transporters belonging to the ATP-Binding Cassette superfamily. These proteins are broadly represented in genomes of both Gram-positive and Gram-negative bacteria and are implicated in virulence and protection against antibiotics and peptide toxins. MacB transporter functions together with MacA, a periplasmic membrane fusion protein, which stimulates MacB ATPase. In Gram-negative bacteria, MacA is believed to couple ATP hydrolysis to transport of substrates across the outer membrane through a TolC-like channel. In this study, we report a real-time analysis of concurrent ATP hydrolysis and assembly of MacAB-TolC complex. MacB binds nucleotides with a low millimolar affinity and fast on- and off-rates. In contrast, MacA-MacB complex is formed with a nanomolar affinity, which further increases in the presence of ATP. Our results strongly suggest that association between MacA and MacB is stimulated by ATP binding to MacB but remains unchanged during ATP hydrolysis cycle. We also found that the large periplasmic loop of MacB plays the major role in coupling reactions separated in two different membranes. This loop is required for MacA-dependent stimulation of MacB ATPase and at the same time, contributes to recruitment of TolC into a trans-envelope complex. PMID:23057817

  3. Fungal secretomes enhance sugar beet pulp hydrolysis

    PubMed Central

    Kracher, Daniel; Oros, Damir; Yao, Wanying; Preims, Marita; Rezic, Iva; Haltrich, Dietmar; Rezic, Tonci; Ludwig, Roland

    2014-01-01

    The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g–1 protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1–17.5 mg g–1 SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load. PMID:24677771

  4. Palm Date Fibers: Analysis and Enzymatic Hydrolysis

    PubMed Central

    Shafiei, Marzieh; Karimi, Keikhosro; Taherzadeh, Mohammad J.

    2010-01-01

    Waste palm dates were subjected to analysis for composition and enzymatic hydrolysis of their flesh fibers. The fruit contained 32% glucose and 30% fructose, while the water-insoluble fibers of its flesh consisted of 49.9% lignin and 20.9% polysaccharides. Water-insoluble fibers were settled to 55% of its initial volume in 12 h. The presence of skin and flesh colloidal fibers results in high viscosity and clogging problems during industrial processes. The settling velocity of the fibers was improved by enzymatic hydrolysis. Hydrolysis resulted in 84.3% conversion of the cellulosic part of the fibers as well as reducing the settling time to 10 minutes and the final settled volume to 4% of the initial volume. It implies easier separation of the fibers and facilitates fermentation processes in the corresponding industries. Two kinds of high- and low-lignin fibers were identified from the water-insoluble fibers. The high-lignin fibers (75% lignin) settled easily, while the low-lignin fibers (41.4% lignin) formed a slurry suspension which settled very slowly. The hydrophilicity of these low-lignin fibers is the major challenge of the industrial processes. PMID:21151438

  5. Fungal secretomes enhance sugar beet pulp hydrolysis.

    PubMed

    Kracher, Daniel; Oros, Damir; Yao, Wanying; Preims, Marita; Rezic, Iva; Haltrich, Dietmar; Rezic, Tonci; Ludwig, Roland

    2014-04-01

    The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g(-1) protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1-17.5 mg g(-1) SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load. PMID:24677771

  6. Peptide Toxins in Solitary Wasp Venoms.

    PubMed

    Konno, Katsuhiro; Kazuma, Kohei; Nihei, Ken-ichi

    2016-04-01

    Solitary wasps paralyze insects or spiders with stinging venom and feed the paralyzed preys to their larva. Accordingly, the venoms should contain a variety of constituents acting on nervous systems. However, only a few solitary wasp venoms have been chemically studied despite thousands of species inhabiting the planet. We have surveyed bioactive substances in solitary wasp venoms found in Japan and discovered a variety of novel bioactive peptides. Pompilidotoxins (PMTXs), in the venoms of the pompilid wasps Anoplius samariensis and Batozonellus maculifrons, are small peptides consisting of 13 amino acids without a disulfide bond. PMTXs slowed Na⁺ channel inactivation, in particular against neuronal type Na⁺ channels, and were rather selective to the Nav1.6 channel. Mastoparan-like cytolytic and antimicrobial peptides are the major components of eumenine wasp venoms. They are rich in hydrophobic and basic amino acids, adopting a α-helical secondary structure, and showing mast cell degranulating, antimicrobial and hemolytic activities. The venom of the spider wasp Cyphononyx fulvognathus contained four bradykinin-related peptides. They are hyperalgesic and, dependent on the structure, differently associated with B₁ or B₂ receptors. Further survey led to the isolation of leucomyosuppressin-like FMRFamide peptides from the venoms of the digger wasps Sphex argentatus and Isodontia harmandi. These results of peptide toxins in solitary wasp venoms from our studies are summarized. PMID:27096870

  7. Peptide Toxins in Solitary Wasp Venoms

    PubMed Central

    Konno, Katsuhiro; Kazuma, Kohei; Nihei, Ken-ichi

    2016-01-01

    Solitary wasps paralyze insects or spiders with stinging venom and feed the paralyzed preys to their larva. Accordingly, the venoms should contain a variety of constituents acting on nervous systems. However, only a few solitary wasp venoms have been chemically studied despite thousands of species inhabiting the planet. We have surveyed bioactive substances in solitary wasp venoms found in Japan and discovered a variety of novel bioactive peptides. Pompilidotoxins (PMTXs), in the venoms of the pompilid wasps Anoplius samariensis and Batozonellus maculifrons, are small peptides consisting of 13 amino acids without a disulfide bond. PMTXs slowed Na+ channel inactivation, in particular against neuronal type Na+ channels, and were rather selective to the Nav1.6 channel. Mastoparan-like cytolytic and antimicrobial peptides are the major components of eumenine wasp venoms. They are rich in hydrophobic and basic amino acids, adopting a α-helical secondary structure, and showing mast cell degranulating, antimicrobial and hemolytic activities. The venom of the spider wasp Cyphononyx fulvognathus contained four bradykinin-related peptides. They are hyperalgesic and, dependent on the structure, differently associated with B1 or B2 receptors. Further survey led to the isolation of leucomyosuppressin-like FMRFamide peptides from the venoms of the digger wasps Sphex argentatus and Isodontia harmandi. These results of peptide toxins in solitary wasp venoms from our studies are summarized. PMID:27096870

  8. Peptic and tryptic hydrolysis of native and heated whey protein to reduce its antigenicity.

    PubMed

    Kim, S B; Ki, K S; Khan, M A; Lee, W S; Lee, H J; Ahn, B S; Kim, H S

    2007-09-01

    incubation of heated WPC with 1% pepsin and then with 1% trypsin was the most effective for producing low-antigenic hydrolysates by WPC hydrolysis and obtaining low molecular weight small peptides. Further research is warranted to identify the low molecular weight small peptides in the WPC hydrolysates produced by pepsin and trypsin, which may enhance the use of whey. PMID:17699020

  9. Endogenous peptide(s) inhibiting [3H]cocaine binding in mouse brain.

    PubMed

    Reith, M E; Sershen, H; Lajtha, A

    1980-12-01

    The supernatant fraction of centrifuged homogenate of brain tissue contains material that inhibits the saturable binding of [3H]cocaine to crude mouse brain membranes. This material was subjected to heat treatment to remove protein; further purification was achieved by filtering through an Amicon UM-10 membrane ultrafilter and gel filtration of the ultrafiltrate on Sephadex G-25. Sensitivity to acid hydrolysis and peptidase action indicates that the inhibitory activity resides in peptide material with low molecular weight. The partially purified inhibitor has similar effects to that of cocaine on the specific binding of various ligands to opiate and nonopiate receptors in mouse brain membranes. PMID:6261176

  10. Effective, Facile, and Selective Hydrolysis of the Chemical Warfare Agent VX Using Zr6-Based Metal-Organic Frameworks.

    PubMed

    Moon, Su-Young; Wagner, George W; Mondloch, Joseph E; Peterson, Gregory W; DeCoste, Jared B; Hupp, Joseph T; Farha, Omar K

    2015-11-16

    The nerve agent VX is among the most toxic chemicals known to mankind, and robust solutions are needed to rapidly and selectively deactivate it. Herein, we demonstrate that three Zr6-based metal-organic frameworks (MOFs), namely, UiO-67, UiO-67-NH2, and UiO-67-N(Me)2, are selective and highly active catalysts for the hydrolysis of VX. Utilizing UiO-67, UiO-67-NH2, and UiO-67-N(Me)2 in a pH 10 buffered solution of N-ethylmorpholine, selective hydrolysis of the P-S bond in VX was observed. In addition, UiO-67-N(Me)2 was found to catalyze VX hydrolysis with an initial half-life of 1.8 min. This half-life is nearly 3 orders of magnitude shorter than that of the only other MOF tested to date for hydrolysis of VX and rivals the activity of the best nonenzymatic materials. Hydrolysis utilizing Zr-based MOFs is also selective and facile in the absence of pH 10 buffer (just water) and for the destruction of the toxic byproduct EA-2192. PMID:26505999

  11. Phosphine and thiophene cyclopalladated complexes: hydrolysis reactions in strong acidic media.

    PubMed

    García, Begoña; Hoyuelos, Francisco J; Ibeas, Saturnino; Muñoz, María S; Navarro, Ana M; Peñacoba, Indalecio A; Leal, José M

    2010-12-01

    The mechanisms for the hydrolysis of organopalladium complexes [Pd(CNN)R]BF(4) (R=P(OPh)(3), PPh(3), and SC(4)H(8)) were investigated at 25 °C by using UV/Vis absorbance measurements in 10 % v/v ethanol/water mixtures containing different sulphuric acid concentrations in the 1.3-11.7 M range. In all cases, a biphasic behavior was observed with rate constants k(1obs), which corresponds to the initial step of the hydrolysis reaction, and k(2obs), where k(1obs)>k(2obs). The plots of k(1obs) and k(2obs) versus sulfuric acid concentration suggest a change in the reaction mechanism. The change with respect to the k(1obs) value corresponds to 35 %, 2 %, and 99 % of the protonated complexes for R=PPh(3), P(OPh)(3), and SC(4)H(8), respectively. Regarding k(2obs), the change occurred in all cases at about 6.5 M H(2)SO(4) and matched up with the results reported for the hydrolysis of the 2-acetylpyridinephenylhydrazone (CNN) ligand. By using the excess acidity method, the mechanisms were elucidated by carefully looking at the variation of k(i),(obs) (i=1,2) versus cH+. The rate-determining constants, k(0,A-1), k(0,A-2), and k(0,A-SE2) were evaluated in all cases. The R=P(OPh)(3) complex was most reactive due to its π-acid character, which favors the rupture of the trans nitrogen-palladium bond in the A-2 mechanism and also that of the pyridine nitrogen-palladium bond in the A-1 mechanism. The organometallic bond exerts no effect on the relative basicity of the complexes, which are strongly reliant on the substituent. PMID:21125574

  12. Angiotensin I Converting enzyme inhibitory peptides from commercial wet- and dry-milled corn germ

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bioprocesses were developed to enhance the value of proteins from de-oiled corn germ. Proteins were hydrolyzed with trypsin, GC106, Flavourzyme or thermolysin in order to free the bioactive peptide sequences. Protein hydrolysis, at an enzyme to substrate ratio of 1:250, was greater for wet- than d...

  13. Chemical Synthesis of Human Insulin-Like Peptide-6.

    PubMed

    Wu, Fangzhou; Mayer, John P; Zaykov, Alexander N; Zhang, Fa; Liu, Fa; DiMarchi, Richard D

    2016-07-01

    Human insulin-like peptide-6 (INSL-6) belongs to the insulin superfamily and shares the distinctive disulfide bond configuration of human insulin. In this report we present the first chemical synthesis of INSL-6 utilizing fluorenylmethyloxycarbonyl-based (Fmoc) solid-phase peptide chemistry and regioselective disulfide bond construction protocols. Due to the presence of an oxidation-sensitive tryptophan residue, two new orthogonal synthetic methodologies were developed. The first method involved the identification of an additive to suppress the oxidation of tryptophan during iodine-mediated S-acetamidomethyl (Acm) deprotection and the second utilized iodine-free, sulfoxide-directed disulfide bond formation. The methodologies presented here offer an efficient synthetic route to INSL-6 and will further improve synthetic access to other multiple-disulfide-containing peptides with oxidation-sensitive residues. PMID:27259101

  14. Brain natriutetic peptide test

    MedlinePlus

    ... medlineplus.gov/ency/article/007509.htm Brain natriuretic peptide test To use the sharing features on this page, please enable JavaScript. Brain natriuretic peptide (BNP) test is a blood test that measures ...

  15. Vasoactive intestinal peptide test

    MedlinePlus

    ... medlineplus.gov/ency/article/003508.htm Vasoactive intestinal peptide test To use the sharing features on this page, please enable JavaScript. Vasoactive intestinal peptide (VIP) is a test that measures the amount ...

  16. Coumarin-decorated Schiff base hydrolysis as an efficient driving force for the fluorescence detection of water in organic solvents.

    PubMed

    Kim, Won Young; Shi, Hu; Jung, Hyo Sung; Cho, Daeheum; Verwilst, Peter; Lee, Jin Yong; Kim, Jong Seung

    2016-07-01

    A coumarin based Schiff base was found to be an excellent indicator of moisture, via rapid in situ hydrolysis. A structure-relationship examination of a small library of Schiff bases revealed the critical importance of hydrogen bond acceptors in close proximity to the imine bond, and this observation was further supported by theoretical calculations as well as the solid state structure analysis. The most sensitive compound demonstrated a limit of detection and quantification of 0.18% and 0.54% v/v water in DMSO, respectively. PMID:27333263

  17. [SYNTHETIC PEPTIDE VACCINES].

    PubMed

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested. PMID:27145593

  18. Using Multiple Bonding Strategies.

    PubMed

    Larson, Thomas D

    2015-01-01

    There are many ways to bond to tooth structure, some micro-mechanical some chemical, some a combination. Different dentin bonding materials have different bonding strengths to differently prepared surfaces, and because of differences in their nature, different areas of tooth structure present peculiar bonding challenges. This paper will review a variety of material types, elucidating their particular bonding strengths and commenting on improved bonding strategies to increase durability, strength, and favorable pulpal response. In this discussion, resin dentin bonding systems, glass ionomers, Gluma, resin cements, and newer combined products will br reviewed. PMID:26485903

  19. An in situ XAS study of ferric iron hydrolysis and precipitation in the presence of perchlorate, nitrate, chloride and sulfate

    NASA Astrophysics Data System (ADS)

    Collins, Richard N.; Rosso, Kevin M.; Rose, Andrew L.; Glover, Chris J.; David Waite, T.

    2016-03-01

    Using a novel combination of in situ potentiometric experiments and quick-scanning XAS we present Fe K-edge XAS spectra (to k = 12 Å-1) during FeIII hydrolysis and precipitation in 0.33 M Fe(ClO4)3, Fe(NO3)3, FeCl3 and Fe2(SO4)3 solutions up to pH 4.8. Edge-sharing FeIII polymers appeared almost immediately upon hydrolysis with strong evidence for a μ-oxo dimer species forming in the Fe(ClO4)3, Fe(NO3)3 and FeCl3 solutions. The effects of SO4 on hydrolysis and polymerization pathways included inhibition of both the formation of the μ-oxo dimer and double corner FeIII bonding, ultimately resulting in the precipitation of schwertmannite. As such, under these experimental conditions, double corner FeIII bonding appears to be critical to the formation of ferrihydrite. The spectral trends indicated that the decomposition/transformation of the dimer was sudden and broadly coincident with shortening average Fe-O bond distances, increased Fe neighbors at ∼3.43 Å and a pre-edge energy transformation suggestive of decreased ligand field strength as well as increasing proportions of tetrahedral FeIII. This result suggests that the incorporation of tetrahedral FeIII into ferrihydrite occurs only at the latter stages of extended polymerization.

  20. PH dependent adhesive peptides

    DOEpatents

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  1. Antimicrobial Peptides in 2014

    PubMed Central

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  2. Antimicrobial peptides in 2014.

    PubMed

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  3. Characterization of specificity of subtilisin Carlsberg towards peptide T by high-performance liquid chromatography and electrospray mass spectrometry.

    PubMed

    Nedjar-Arroumea; Paon; Koralewski; Friboulet; Kouach; Briand; Leroy; Guillochon

    2000-03-01

    Peptide T has a sequence (Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr) belonging to HIV envelope that is involved in the interaction with CD(4) receptor of T lymphocytes. Research of protease activities towards this peptide is very relevant for AIDS therapy. Characterization of specificity of subtilisin Carlsberg towards this very hydrophilic peptide is proposed by using high-performance liquid chromatography and mass spectrometry. Peptide T was totally hydrolysed by the protease after 24 h. Separation of hydrophilic fragments was perfected with an hydrophilic stationary phase and a reversed acetonitrile gradient. Peptide masses were determined by ion spray mass spectrometry. Four primary and one secondary hydrolysis products were found, corresponding to cleavage at the carboxylic side of threonine. Specifities of subtilisin Carlsberg towards the Segments 19 to 26 of bovine pancreatic ribonuclease A, an homologous fragment of peptide T, and peptide T were compared. PMID:10713210

  4. Hierarchical organization of ferrocene-peptides.

    PubMed

    Beheshti, Samaneh; Martić, Sanela; Kraatz, Heinz-Bernhard

    2012-07-16

    Hierarchical self-assembly of disubstituted ferrocene (Fc)-peptide conjugates that possess Gly-Val-Phe and Gly-Val-Phe-Phe peptide substituents leads to the formation of nano- and micro-sized assemblies. Hydrogen-bonding and hydrophobic interactions provide directionality to the assembly patterns. The self-assembling behavior of these compounds was studied in solution by using (1)H NMR and circular dichroism (CD) spectroscopies. In the solid state, attenuated total reflectance (ATR) FTIR spectroscopy, single-crystal X-ray diffraction (XRD), powder X-ray diffraction (PXRD), and scanning electron microscopy (SEM) methods were used. Spontaneous self-assembly of Fc-peptides through intra- and intermolecular hydrogen-bonding interactions induces supramolecular assemblies, which further associate and give rise to fibers, large fibrous crystals, and twisted ropes. In the case of Fc[CO-Gly-Val-Phe-OMe](2) (1), molecules initially interact to form pleated sheets that undergo association into long fibers that form bundles and rectangular crystalline cuboids. Molecular offsets and defects, such as screw dislocations and solvent effects that occur during crystal growth, induce the formation of helical arrangements, ultimately leading to large twisted ropes. By contrast, the Fc-tetrapeptide conjugate Fc[CO-Gly-Val-Phe-Phe-OMe](2) (2) forms a network of nanofibers at the supramolecular level, presumably due to the additional hydrogen-bonding and hydrophobic interactions that stem from the additional Phe residues. PMID:22707407

  5. Biocatalytic conversion of poultry processing leftovers: Optimization of hydrolytic conditions and peptide hydrolysate characterization.

    PubMed

    Nikolaev, I V; Sforza, S; Lambertini, F; Ismailova, D Yu; Khotchenkov, V P; Volik, V G; Dossena, A; Popov, V O; Koroleva, O V

    2016-04-15

    Peptide hydrolysate (PH) was produced by deep controllable bioconversion of poultry processing leftovers (broiler necks), by means of a multienzyme composition, containing four commercially available enzyme preparations (Alcalase, Neutrase, Flavourzyme, Protamex). The design of multienzyme composition (MEC) was applied to yield a hydrolysate with adjusted properties, including minimized antigenicity and bitterness. The protein recovery was optimized using Box-Behnken response surface design. The individual and interactive effects of hydrolysis conditions (time, hydromodule and MEC dosage) were studied. The experimental data were analyzed by ANOVA method and a well-predictive, second order polynomial model was developed using multiple regression analysis. Optimal hydrolysis conditions were found to be: hydrolysis time 3 h, hydromodule 2.25 l/kg and dosage of MEC 0.25%. The corresponding predicted value for protein recovery was 75.34%, 2 times higher compared to traditional long-term heating hydrolysis. The PH obtained is a low allergenic product with high antioxidant capacity. PMID:26616995

  6. Oxygen radical-mediated oxidation reactions of an alanine peptide motif - density functional theory and transition state theory study

    PubMed Central

    2012-01-01

    Background Oxygen-base (O-base) oxidation in protein backbone is important in the protein backbone fragmentation due to the attack from reactive oxygen species (ROS). In this study, an alanine peptide was used model system to investigate this O-base oxidation by employing density functional theory (DFT) calculations combining with continuum solvent model. Detailed reaction steps were analyzed along with their reaction rate constants. Results Most of the O-base oxidation reactions for this alanine peptide are exothermic except for the bond-breakage of the Cα-N bond to form hydroperoxy alanine radical. Among the reactions investigated in this study, the activated energy of OH α-H abstraction is the lowest one, while the generation of alkylperoxy peptide radical must overcome the highest energy barrier. The aqueous situation facilitates the oxidation reactions to generate hydroxyl alanine peptide derivatives except for the fragmentations of alkoxyl alanine peptide radical. The Cα-Cβ bond of the alkoxyl alanine peptide radical is more labile than the peptide bond. Conclusion the rate-determining step of oxidation in protein backbone is the generation of hydroperoxy peptide radical via the reaction of alkylperoxy peptide radical with HO2. The stabilities of alkylperoxy peptide radical and complex of alkylperoxy peptide radical with HO2 are crucial in this O-base oxidation reaction. PMID:22524792

  7. Microbial degradation rates of small peptides and amino acids in the oxygen minimum zone of Chilean coastal waters

    NASA Astrophysics Data System (ADS)

    Pantoja, Silvio; Rossel, Pamela; Castro, Rodrigo; Cuevas, L. Antonio; Daneri, Giovanni; Córdova, Candy

    2009-07-01

    We found similar microbial degradation rates of labile dissolved organic matter in oxic and suboxic waters off northern Chile. Rates of peptide hydrolysis and amino acid uptake in unconcentrated water samples were not low in the water column where oxygen concentration was depleted. Hydrolysis rates ranged from 65 to 160 nmol peptide L -1 h -1 in the top 20 m, 8-28 nmol peptide L -1 h -1 between 100 and 300 m (O 2-depleted zone), and 14-19 nmol peptide L -1 h -1 between 600 and 800 m. Dissolved free amino acid uptake rates were 9-26, 3-17, and 6 nmol L -1 h -1 at similar depth intervals. Since these findings are consistent with a model of comparable potential activity of microbes in degrading labile substrates of planktonic origin, we suggest, as do other authors, that differences in decomposition rates with high and low oxygen concentrations may be a matter of substrate lability. The comparison between hydrolysis and uptake rates indicates that microbial peptide hydrolysis occurs at similar or faster rates than amino acid uptake in the water column, and that the hydrolysis of peptides is not a rate-limiting step for the complete remineralization of labile macromolecules. Low O 2 waters process about 10 tons of peptide carbon per h, double the amount processed in surface-oxygenated water. In the oxygen minimum zone, we suggest that the C balance may be affected by the low lability of the dissolved organic matter when this is upwelled to the surface. An important fraction of dissolved organic matter is processed in the oxygen minimum layer, a prominent feature of the coastal ocean in the highly productive Humboldt Current System.

  8. Hydrolysis of ferric chloride in solution

    SciTech Connect

    Lussiez, G.; Beckstead, L.

    1996-11-01

    The Detox{trademark} process uses concentrated ferric chloride and small amounts of catalysts to oxidize organic compounds. It is under consideration for oxidizing transuranic organic wastes. Although the solution is reused extensively, at some point it will reach the acceptable limit of radioactivity or maximum solubility of the radioisotopes. This solution could be cemented, but the volume would be increased substantially because of the poor compatibility of chlorides and cement. A process has been developed that recovers the chloride ions as HCl and either minimizes the volume of radioactive waste or permits recycling of the radioactive chlorides. The process involves a two-step hydrolysis at atmospheric pressure, or preferably under a slight vacuum, and relatively low temperature, about 200{degrees}C. During the first step of the process, hydrolysis occurs according to the reaction below: FeCl{sub 3 liquid} + H{sub 2}O {r_arrow} FeOCl{sub solid} + 2 HCl{sub gas} During the second step, the hot, solid, iron oxychloride is sprayed with water or placed in contact with steam, and hydrolysis proceeds to the iron oxide according to the following reaction: 2 FeOCl{sub solid} + H{sub 2}O {r_arrow} Fe{sub 2}O{sub 3 solid} + 2 HCl{sub gas}. The iron oxide, which contains radioisotopes, can then be disposed of by cementation or encapsulation. Alternately, these chlorides can be washed off of the solids and can then either be recycled or disposed of in some other way.

  9. Active-site amino acid residues in γ-glutamyltransferase and the nature of the γ-glutamyl-enzyme bond

    PubMed Central

    Elce, John S.

    1980-01-01

    Active-site residues in rat kidney γ-glutamyltransferase (EC 2.3.2.2) were investigated by means of chemical modification. 1. In the presence of maleate, the activity was inhibited by phenylmethanesulphonyl fluoride, and the inhibition was not reversed by β-mercaptoethanol, suggesting that a serine residue is close to the active site, but is shielded except in the presence of maleate. 2. Treatment of the enzyme with N-acetylimidazole modified an amino group, exposed a previously inaccessible cysteine residue and inhibited hydrolysis of the γ-glutamyl-enzyme intermediate, but not its formation. 3. After reaction of the enzyme successively with N-acetylimidazole and with non-radioactive iodoacetamide/serine/borate, two active-site residues reacted with iodo[14C]acetamide. One of these possessed a carboxy group, which formed a [14C]glycollamide ester, and the other was cysteine, shown by isolation of S-[14C]carboxymethylcysteine after acid hydrolysis. When N-acetylimidazole treatment was omitted, only the carboxy group reacted with iodo[14C]acetamide. 4. Isolation of the γ-[14C]glutamyl-enzyme intermediate was made easier by prior treatment of the enzyme with N-acetylimidazole. The γ-glutamyl-enzyme bond was stable to performic acid, and to hydroxylamine/urea at pH10, but was hydrolysed slowly at pH12, indicating attachment of the γ-[14C]glutamyl group in amide linkage to an amino group on the enzyme. Proteolysis of the γ-[14C]glutamyl-enzyme after performic acid oxidation gave rise to a small acidic radioactive peptide that was resistant to further proteolysis and was not identical with γ-glutamyl-ε-lysine. 5. A scheme for the catalytic mechanism is proposed. PMID:6104953

  10. Peptide folding driven by Van der Waals interactions.

    PubMed

    Sung, Shen-Shu

    2015-09-01

    Contrary to the widespread view that hydrogen bonding and its entropy effect play a dominant role in protein folding, folding into helical and hairpin-like structures is observed in molecular dynamics (MD) simulations without hydrogen bonding in the peptide-solvent system. In the widely used point charge model, hydrogen bonding is calculated as part of the interaction between atomic partial charges. It is removed from these simulations by setting atomic charges of the peptide and water to zero. Because of the structural difference between the peptide and water, van der Waals (VDW) interactions favor peptide intramolecular interactions and are a major contributing factor to the structural compactness. These compact structures are amino acid sequence dependent and closely resemble standard secondary structures, as a consequence of VDW interactions and covalent bonding constraints. Hydrogen bonding is a short range interaction and it locks the approximate structure into the specific secondary structure when it is included in the simulation. In contrast to standard molecular simulations where the total energy is dominated by charge-charge interactions, these simulation results will give us a new view of the folding mechanism. PMID:26013298

  11. The Alkaline Hydrolysis of Sulfonate Esters: Challenges in Interpreting Experimental and Theoretical Data

    PubMed Central

    2013-01-01

    Sulfonate ester hydrolysis has been the subject of recent debate, with experimental evidence interpreted in terms of both stepwise and concerted mechanisms. In particular, a recent study of the alkaline hydrolysis of a series of benzene arylsulfonates (Babtie et al., Org. Biomol. Chem.10, 2012, 8095) presented a nonlinear Brønsted plot, which was explained in terms of a change from a stepwise mechanism involving a pentavalent intermediate for poorer leaving groups to a fully concerted mechanism for good leaving groups and supported by a theoretical study. In the present work, we have performed a detailed computational study of the hydrolysis of these compounds and find no computational evidence for a thermodynamically stable intermediate for any of these compounds. Additionally, we have extended the experimental data to include pyridine-3-yl benzene sulfonate and its N-oxide and N-methylpyridinium derivatives. Inclusion of these compounds converts the Brønsted plot to a moderately scattered but linear correlation and gives a very good Hammett correlation. These data suggest a concerted pathway for this reaction that proceeds via an early transition state with little bond cleavage to the leaving group, highlighting the care that needs to be taken with the interpretation of experimental and especially theoretical data. PMID:24279349

  12. Mechanistic studies of the base-catalyzed hydrolysis of pyridine nucleotides

    SciTech Connect

    Johnson, R.W.; Marschner, T.M.; Malver, O.; Sleath, P.R.; Oppenheimer, N.J.

    1986-05-01

    The pH dependence of base-catalyzed hydrolysis of ..beta..-NAD has been determined over the range from pH 8.5 to 13.5. Below pH 10.5 the reaction rate constant is linearly dependent on hydroxide concentration whereas above pH 12.5 the reaction becomes pH independent. A nonlinear least squares fit of the data yields a pK/sub a/ of 12.2, corresponding to the ionization of the 2'-OH of the nicotinamide ribose as determined by /sup 1/H and /sup 13/C NMR. Based on these data, as well as solvent isotope effects and data from previous investigators, the authors propose that ionization of the ribose diol stabilizes an oxonium ion intermediate, thus, facilitating S/sub N/1 hydrolysis of the nicotinamide-glycosyl bond with release of nicotinamide. Further evidence for this mechanism is provided by investigation of the 2',3'-O-isopropylidine nicotinamide riboside. This compound is found to be highly resistant to hydrolysis in base and product analysis by NMR reveals that only 2-hydroxy-3-pyridinecarboxaldehyde is released. The influence on the reaction rate and mechanism resulting from other modifications of the sugar moiety of nicotinamide nucleosides are discussed.

  13. Synergistic effect between defect sites and functional groups on the hydrolysis of cellulose over activated carbon.

    PubMed

    Foo, Guo Shiou; Sievers, Carsten

    2015-02-01

    The chemical oxidation of activated carbon by H2 O2 and H2 SO4 is investigated, structural and chemical modifications are characterized, and the materials are used as catalysts for the hydrolysis of cellulose. Treatment with H2 O2 enlarges the pore size and imparts functional groups such as phenols, lactones, and carboxylic acids. H2 SO4 treatment targets the edges of carbon sheets primarily, and this effect is more pronounced with a higher temperature. Adsorption isotherms demonstrate that the adsorption of oligomers on functionalized carbon is dominated by van der Waals forces. The materials treated chemically are active for the hydrolysis of cellulose despite the relative weakness of most of their acid sites. It is proposed that a synergistic effect between defect sites and functional groups enhances the activity by inducing a conformational change in the glucan chains if they are adsorbed at defect sites. This activates the glycosidic bonds for hydrolysis by in-plane functional groups. PMID:25504913

  14. Glycosyl conformational and inductive effects on the acid catalysed hydrolysis of purine nucleosides.

    PubMed Central

    Jordan, F; Niv, H

    1977-01-01

    The log kobs vs. pH profiles were determined in the intermediate acidity region for the glycosyl hydrolysis of guanosine and its 8-amino, 8-monomethylamino, 8-dimethylamino and 8-bromo derivatives. The decreased rate of the 8-amino and enhanced rate of the 8-bromo compound compared to guanosine support an A type mechanism: base protonation followed by glycosyl bond cleavage. All three 8-amino guanosines exhibited log kobs - pH profiles clearly showing that both mono and di-base protonated nucleosides undergo hydrolysis. The 700 fold rate acceleration of 8-N(CH3)-guanosine compared to 8-NHCH3-guanosine and the 110 fold rate acceleration of 8-N(CH3)2-adenosine compared to 8-NHCH3-adenosine could be unequivocally attributed to the fixed syn glycosyl conformation of both 8-dimethylamino compounds and relief of steric compression upon hydrolysis in these molecules. The lack of anomerization of all substrates during the course of the reaction supports an A rather than a Schiff-base mechanism. PMID:17100

  15. Insights into the Chemomechanical Coupling of the Myosin Motor from Simulation of Its ATP Hydrolysis Mechanism

    SciTech Connect

    Schwarzl, S.M.; Smith, Jeremy C; Fischer, S.

    2006-03-01

    The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP{circle_dot}Pi state of nucleotide-bound myosin.

  16. Improved method for detection of starch hydrolysis

    SciTech Connect

    Ohawale, M.R.; Wilson, J.J.; Khachatourians, G.G.; Ingledew, W.M.

    1982-09-01

    A new starch hydrolysis detection method which does not rely on iodine staining or the use of color-complexed starch is described. A linear relationship was obtained with agar-starch plates when net clearing zones around colonies of yeasts were plotted against enzyme levels (semilogarithm scale) produced by the same yeast strains in liquid medium. A similar relationship between starch clearing zones and alpha-amylase levels from three different sources was observed. These observations suggest that the method is useful in mutant isolations, strain improvement programs, and the prediction of alpha-amylase activities in culture filtrates or column effluents. (Refs. 18).

  17. Pretreatment and enzymatic hydrolysis of lignocellulosic biomass

    NASA Astrophysics Data System (ADS)

    Corredor, Deisy Y.

    The performance of soybean hulls and forage sorghum as feedstocks for ethanol production was studied. The main goal of this research was to increase fermentable sugars' yield through high-efficiency pretreatment technology. Soybean hulls are a potential feedstock for production of bio-ethanol due to their high carbohydrate content (≈50%) of nearly 37% cellulose. Soybean hulls could be the ideal feedstock for fuel ethanol production, because they are abundant and require no special harvesting and additional transportation costs as they are already in the plant. Dilute acid and modified steam-explosion were used as pretreatment technologies to increase fermentable sugars yields. Effects of reaction time, temperature, acid concentration and type of acid on hydrolysis of hemicellulose in soybean hulls and total sugar yields were studied. Optimum pretreatment parameters and enzymatic hydrolysis conditions for converting soybean hulls into fermentable sugars were identified. The combination of acid (H2SO4, 2% w/v) and steam (140°C, 30 min) efficiently solubilized the hemicellulose, giving a pentose yield of 96%. Sorghum is a tropical grass grown primarily in semiarid and dry parts of the world, especially in areas too dry for corn. The production of sorghum results in about 30 million tons of byproducts mainly composed of cellulose, hemicellulose, and lignin. Forage sorghum such as brown midrib (BMR) sorghum for ethanol production has generated much interest since this trait is characterized genetically by lower lignin concentrations in the plant compared with conventional types. Three varieties of forage sorghum and one variety of regular sorghum were characterized and evaluated as feedstock for fermentable sugar production. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and X-Ray diffraction were used to determine changes in structure and chemical composition of forage sorghum before and after pretreatment and enzymatic hydrolysis

  18. /sup 18/O isotope effect in /sup 13/C nuclear magnetic resonance spectroscopy. Part 9. Hydrolysis of benzyl phosphate by phosphatase enzymes and in acidic aqueous solutions

    SciTech Connect

    Parente, J.E.; Risley, J.M.; Van Etten, R.L.

    1984-12-26

    The /sup 18/O isotope-induced shifts in /sup 13/C and /sup 31/P nuclear magnetic resonance (NMR) spectroscopy were used to establish the position of bond cleavage in the phosphatase-catalyzed and acid-catalyzed hydrolysis reactions of benzyl phosphate. The application of the /sup 18/O-isotope effect in NMR spectroscopy affords a continuous, nondestructive assay method for following the kinetics and position of bond cleavage in the hydrolytic process. The technique provides advantages over most discontinuous methods in which the reaction components must be isolated and converted to volatile derivatives prior to analysis. In the present study, (..cap alpha..-/sup 13/C,ester-/sup 18/O)benzyl phosphate and (ester-/sup 18/O)benzyl phosphate were synthesized for use in enzymatic and nonenzymatic studies. Hydrolysis reactions catalyzed by the alkaline phosphatase from E. coli and by the acid phosphatases isolated from human prostate and human liver were all accompanied by cleavage of the substrate phosphorus-oxygen bond consistent with previously postulated mechanisms involving covalent phosphoenzyme intermediates. An extensive study of the acid-catalyzed hydrolysis of benzyl phosphate at 75/sup 0/C revealed that the site of bond cleavage is dependent on pH. At pH less than or equal to 1.3, the hydrolysis proceeds with C-O bond cleavage; at 1.3 < pH < 2.0, there is a mixture of C-O and P-O bond scission, the latter progressively predominating as the pH is raised; at pH greater than or equal to 2.0, the hydrolysis proceeds with exclusive P-O bond scission. (S)-(+)-(..cap alpha..-/sup 2/H)Benzyl phosphate was also synthesized. Hydrolysis of this chiral benzyl derivative demonstrated that the acid-catalyzed C-O bond scission of benzyl phosphate proceeds by an A-1 (S/sub N/1) mechanism with 70% racemization and 30% inversion at carbon. 37 references, 4 figures, 2 tables.

  19. One-step surface modification of polyurethane using affinity binding peptides for enhanced fouling resistance.

    PubMed

    Wang, Yibing; Yu, Yong; Zhang, Liting; Qin, Peng; Wang, Ping

    2015-01-01

    Affinity binding peptides were examined for surface fabrication of synthetic polymeric materials. Peptides possessing strong binding affinities toward polyurethane (PU) were discovered via biopanning of M13 phage peptide library. The apparent binding constant (K(app)) was as high as 2.68 × 10(9) M(-1) with surface peptide density exceeded 1.8 μg/cm(2). Structural analysis showed that the ideal peptide had a high content (75%) of H-donor amino acid residues, and that intensified hydrogen bond interaction was the key driving force for the highly stable binding of peptides on PU. PU treated with such affinity peptides promises applications as low-fouling materials, as peptides increased its wettability and substantially reduced protein adsorption and cell adhesion. These results demonstrated a facile but highly efficient one-step strategy for surface property modification of polymeric materials for biotechnological applications. PMID:25732121

  20. Hydrolysis of protein and model dipeptide substrated by attached and nonattached marine Pseudomonas sp. strain NCIMB 2021

    SciTech Connect

    Griffith, P.C.; Fletcher, M. )

    1991-08-01

    Rates of substrate hydrolysis by nonattached bacteria and by bacteria attached to particles derived from marine diatom frustules were estimated by using two substrates, a dipeptide analog and a protein. Adsorption of the two substrates onto the particles was also evaluated. Methyl-coumarinyl-amide-leucine (MCA-leucine) was used to estimate hydrolysis of dipeptides by measuring an increase in fluorescence as MCA-leucine was hydrolyzed to leucine and the fluorochrome methylcoumarin. To examine hydrolysis of a larger molecule, was prepared a radiolabeled protein by {sup 14}C-methylation of bovine serum albumin. The rate of protein hydrolysis in samples of particle-attached or nonattached bacteria was estimated by precipitating all nonhydrolyzed protein with cold trichloroacetic acid and then determining the trichloroacetic acid-soluble radiolabeled material, which represented methyl-{sup 14}C-peptides and -amino acids. About 25% of the MCA-leucine adsorbed to the particles. MCA-leucine was hydrolyzed faster by nonattached than attached bacteria, which was probably related to its tendency to remain dissolved in the liquid phase. In contrast, almost 100% of the labeled protein adsorbed to the particles. Accordingly, protein was much less available to nonattached bacteria but was rapidly hydrolyzed by attached bacteria.

  1. Theoretical study of the alkaline hydrolysis of an aza-β-lactam derivative of clavulanic acid

    NASA Astrophysics Data System (ADS)

    Garcías, Rafael C.; Coll, Miguel; Donoso, Josefa; Muñoz, Francisco

    2003-04-01

    DFT calculations based on the hybrid functional B3LYP/6-31+G * were used to study the alkaline hydrolysis of an aza-clavulanic acid, which results from the substitution of the carbon atom at position 6 in clavulanic acid by a nitrogen atom. The presence of the nitrogen atom endows the compound with special properties; in fact, once formed, the tetrahedral intermediate can evolve with cleavage of the N 4-C 7 or N 6-C 7 bond, which obviously leads to different reaction products. These differential bond cleavages may play a central role in the inactivation of β-lactamases, so the compound may be a powerful inactivator of these enzymes.

  2. Hydrolysis of lanthanide dicarbides: Rates of reaction of cubic and tetragonal solid solutions with water

    SciTech Connect

    McColm, I.J. )

    1993-05-01

    Two series of solid solutions, Ho[sub 1[minus]x]La[sub x]C[sub 2] and Nd[sub 1[minus]x]LaC[sub 2], have been made and their X-ray unit cell parameters measured. The Ho[sub 1[minus]x]La[sub x]C[sub 2] series contains two tetragonal phases and a cubic solid solution series which has enabled the reaction rate constants for the water hydrolysis reaction of a cubic dicarbide phase to be determined for the first time. By comparing the linear rate constants and the activation energies across the two series the nature of bonding in general and the structure of the cubic phase are elucidated. A comparison with microhardness data is made and the change in M-C[sub 2] bonding as a function of composition is considered. 10 refs., 9 figs., 3 tabs.

  3. 29 CFR 2580.412-20 - Use of existing bonds, separate bonds and additional bonding.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 9 2010-07-01 2010-07-01 false Use of existing bonds, separate bonds and additional..., separate bonds and additional bonding. (a) Additional bonding. Section 13 neither prevents additional... or separate bond. (b) Use of existing bonds. Insofar as a bond currently in use is adequate to...

  4. Hydroxide-catalyzed bonding

    NASA Technical Reports Server (NTRS)

    Gwo, Dz-Hung (Inventor)

    2003-01-01

    A method of bonding substrates by hydroxide-catalyzed hydration/dehydration involves applying a bonding material to at least one surface to be bonded, and placing the at least one surface sufficiently close to another surface such that a bonding interface is formed between them. A bonding material of the invention comprises a source of hydroxide ions, and may optionally include a silicate component, a particulate filling material, and a property-modifying component. Bonding methods of the invention reliably and reproducibly provide bonds which are strong and precise, and which may be tailored according to a wide range of possible applications. Possible applications for bonding materials of the invention include: forming composite materials, coating substrates, forming laminate structures, assembly of precision optical components, and preparing objects of defined geometry and composition. Bonding materials and methods of preparing the same are also disclosed.

  5. Template-Directed Ligation of Peptides to Oligonucleotides

    NASA Technical Reports Server (NTRS)

    Bruick, Richard K.; Dawson, Philip E.; Kent, Stephen BH; Usman, Nassim; Joyce, Gerald F.

    1996-01-01

    Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template.

  6. Conformational Restriction of Peptides Using Dithiol Bis-Alkylation.

    PubMed

    Peraro, L; Siegert, T R; Kritzer, J A

    2016-01-01

    Macrocyclic peptides are highly promising as inhibitors of protein-protein interactions. While many bond-forming reactions can be used to make cyclic peptides, most have limitations that make this chemical space challenging to access. Recently, a variety of cysteine alkylation reactions have been used in rational design and library approaches for cyclic peptide discovery and development. We and others have found that this chemistry is versatile and robust enough to produce a large variety of conformationally constrained cyclic peptides. In this chapter, we describe applications, methods, mechanistic insights, and troubleshooting for dithiol bis-alkylation reactions for the production of cyclic peptides. This method for efficient solution-phase macrocyclization is highly useful for the rapid production and screening of loop-based inhibitors of protein-protein interactions. PMID:27586339

  7. Metabolism of Peptide Reporters in Cell Lysates and Single Cells

    PubMed Central

    Proctor, Angela; Wang, Qunzhao; Lawrence, David S.; Allbritton, Nancy L.

    2013-01-01

    The stability of an Abl kinase substrate peptide in a cytosolic lysate and in single cells was characterized. In the cytosolic lysate, the starting peptide was metabolized at an average initial rate of 1.7 ± 0.3 zmol pg−;1 s−;1 with a t1/2 of 1.3 min. Five different fragments formed over time; however, a dominant cleavage site was identified. Multiple rational design cycles were utilized to develop a lead peptide with a phenylalanine and alanine replaced by an (N-methyl)phenylalanine and isoleucine, respectively, to attain cytosolic peptidase resistance while maintaining Abl substrate efficacy. This lead peptide possessed a 15-fold greater lifetime in the cytosolic lysate while attaining a 7-fold improvement in kcat as an Abl kinase substrate compared to the starting peptide. However, when loaded into single cells, the starting peptide and lead peptide possessed nearly identical degradation rates and an altered pattern of fragmentation relative to that in cell lysates. Preferential accumulation of a fragment with cleavage at an Ala-Ala bond in single cells suggested that dissimilar peptidases act on the peptides in the lysate versus single cells. A design strategy for peptide stabilization, analogous to that demonstrated for the lysate, should be effective for stabilization in single cells. PMID:22314840

  8. Kinetic studies of phosphoester hydrolysis promoted by a dimeric tetrazirconium(iv) Wells-Dawson polyoxometalate.

    PubMed

    Luong, Thi Kim Nga; Shestakova, Pavletta; Parac-Vogt, Tatjana N

    2016-07-26

    The catalytic hydrolysis of a phosphoester bond in the DNA-model substrate 4-nitrophenyl phosphate (NPP) promoted by Zr(iv)-substituted Wells-Dawson Na14[Zr4(P2W16O59)2(μ3-O)2(OH)2(H2O)4]·57H2O polyoxometalate (ZrWD 4 : 2) was followed by means of (1)H and (31)P NMR spectroscopy. The hydrolytic reaction proceeded with a rate constant of 8.44 (±0.36) × 10(-5) s(-1) at pD 6.4 and 50 °C, representing a 300-fold rate enhancement in comparison with the spontaneous hydrolysis of NPP (kobs = 2.81 (±0.25) × 10(-7) s(-1)) under the same reaction conditions. The ZrWD 4 : 2 was also active towards hydrolysis of bis(4-nitrophenyl) phosphate (BNPP) and the RNA model substrate 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP). The pD dependence of kobs shows that the rate constants for NPP hydrolysis decrease significantly when the pD values of the reaction mixtures increase. The formation constant (Kf = 190 M(-1)) and catalytic rate constant (kc = 6.40 × 10(-4) s(-1)) for the NPP-ZrWD 4 : 2 complex, activation energy (Ea) of 110.15 ± 7.06 kJ mol(-1), enthalpy of activation (ΔH(‡)) of 109.03 ± 6.86 kJ mol(-1), entropy of activation (ΔS(‡)) of 15.20 ± 2.49 J mol(-1) K(-1), and Gibbs activation energy (ΔG(‡)) of 104.32 ± 6.09 kJ mol(-1) at 37 °C were calculated from kinetic studies. The recyclability of ZrWD 4 : 2 was examined by adding an extra amount (5.0 mM) of NPP twice to a fully hydrolyzed mixture of 5.0 mM NPP and 1.0 mM ZrWD 4 : 2. The interaction between ZrWD 4 : 2 and the P-O bond of NPP was evidenced by a change in the (31)P chemical shift of the (31)P atom in NPP upon addition of ZrWD 4 : 2. Based on (31)P NMR experiments and the kinetic studies, a mechanism for NPP hydrolysis promoted by ZrWD 4 : 2 has been proposed. PMID:27406623

  9. The Hydrolysis of Carbonyl Sulfide at Low Temperature: A Review

    PubMed Central

    Zhao, Shunzheng; Yi, Honghong; Tang, Xiaolong; Jiang, Shanxue; Gao, Fengyu; Zhang, Bowen; Zuo, Yanran; Wang, Zhixiang

    2013-01-01

    Catalytic hydrolysis technology of carbonyl sulfide (COS) at low temperature was reviewed, including the development of catalysts, reaction kinetics, and reaction mechanism of COS hydrolysis. It was indicated that the catalysts are mainly involved metal oxide and activated carbon. The active ingredients which can load on COS hydrolysis catalyst include alkali metal, alkaline earth metal, transition metal oxides, rare earth metal oxides, mixed metal oxides, and nanometal oxides. The catalytic hydrolysis of COS is a first-order reaction with respect to carbonyl sulfide, while the reaction order of water changes as the reaction conditions change. The controlling steps are also different because the reaction conditions such as concentration of carbonyl sulfide, reaction temperature, water-air ratio, and reaction atmosphere are different. The hydrolysis of carbonyl sulfide is base-catalyzed reaction, and the force of the base site has an important effect on the hydrolysis of carbonyl sulfide. PMID:23956697

  10. Peptides@mica: from affinity to adhesion mechanism.

    PubMed

    Gladytz, A; John, T; Gladytz, T; Hassert, R; Pagel, M; Risselada, H J; Naumov, S; Beck-Sickinger, A G; Abel, B

    2016-09-14

    Investigating the adsorption of peptides on inorganic surfaces, on the molecular level, is fundamental for medicinal and analytical applications. Peptides can be potent as linkers between surfaces and living cells in biochips or in implantation medicine. Here, we studied the adsorption process of the positively charged pentapeptide RTHRK, a recently identified binding sequence for surface oxidized silicon, and novel analogues thereof to negatively charged mica surfaces. Homogeneous formation of monolayers in the nano- and low micromolar peptide concentration range was observed. We propose an alternative and efficient method to both quantify binding affinity and follow adhesion behavior. This method makes use of the thermodynamic relationship between surface coverage, measured by atomic force microscopy (AFM), and the concomitant free energy of adhesion. A knowledge-based fit to the autocorrelation of the AFM images was used to correct for a biased surface coverage introduced by the finite lateral resolution of the AFM. Binding affinities and mechanisms were further explored by large scale molecular dynamics (MD) simulations. The combination of well validated MD simulations with topological data from AFM revealed a better understanding of peptide adsorption processes on the atomistic scale. We demonstrate that binding affinity is strongly determined by a peptide's ability to form salt bridges and hydrogen bonds with the surface lattice. Consequently, differences in hydrogen bond formation lead to substantial differences in binding affinity despite conservation of the peptide's overall charge. Further, MD simulations give access to relative changes in binding energy of peptide variations in comparison to a lead compound. PMID:27491508

  11. ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS. II. ACID AND GENERAL BASE CATALYZED HYDROLYSIS

    EPA Science Inventory

    SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to calculate acid and neutral hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition states of a ...

  12. Technical bases for precipitate hydrolysis process operating parameters

    SciTech Connect

    Bannochie, C.J.; Lambert, D.P.

    1992-11-09

    This report provides the experimental data and rationale in support of the operating parameters for tetraphenylborate precipitate hydrolysis specified in WSRC-RP-92-737. The report is divided into two sections, the first dealing with lab-scale precipitate hydrolysis experimentation while the second part addresses large-scale runs conducted to demonstrate the revised operating parameters in the Precipitate Hydrolysis Experimental Facility (PHEF). The program was in conjunction with reducing the nitrite ion level in DWPF feed.

  13. Technical bases for precipitate hydrolysis process operating parameters. Revision 1

    SciTech Connect

    Bannochie, C.J.; Lambert, D.P.

    1992-11-09

    This report provides the experimental data and rationale in support of the operating parameters for tetraphenylborate precipitate hydrolysis specified in WSRC-RP-92-737. The report is divided into two sections, the first dealing with lab-scale precipitate hydrolysis experimentation while the second part addresses large-scale runs conducted to demonstrate the revised operating parameters in the Precipitate Hydrolysis Experimental Facility (PHEF). The program was in conjunction with reducing the nitrite ion level in DWPF feed.

  14. Simulation of continuous and batch hydrolysis of willow

    SciTech Connect

    Zacchi, G.; Dahlbom, J.; Scott, C.D.

    1986-01-01

    The influence of product and enzyme concentrations on the kinetics of the enzymic hydrolysis of alkali-pretreated willow is studied. The hydrolysis was performed in a UF-membrane reactor in which the product concentration was kept constant. An empirical 4-parameter rate equation that gives a good correlation to both continuous and batch hydrolysis data is presented. The model comprises the effects of enzyme concentration and product inhibition. (Refs. 11).

  15. The hydrolysis activity of Adenosine triphosphate in myosin: a theoretical analysis of anomeric effects and the nature of transition state

    PubMed Central

    Yang, Yang; Cui, Qiang

    2009-01-01

    Combined quantum mechanical/molecular mechanical (QM/MM) calculations with density functional theory are employed to analyze two issues related to the hydrolysis activity of Adenosine triphosphate (ATP) in myosin. First, we compare the geometrical properties and electronic structure of ATP in the open (post-rigor) and closed (pre-powerstroke) active sites of the myosin motor domain. Compared to both solution and the open active-site cases, the scissile Pγ-O3β bond of ATP in the closed active-site is shown to be substantially elongated. Natural Bond Orbital (NBO) analysis clearly shows that this structural feature is correlated with the stronger anomeric effects in the closed active site, which involve charge transfers from the lone-pairs in the non-bridging oxygen in the γ phosphate to the anti-bonding orbital of the scissile bond. However, an energetic analysis finds that the ATP molecule is not significantly destabilized by the Pγ-O3β bond elongation. Therefore, despite the notable perturbations in the geometry and electronic structure of ATP as its environment changes from solution to the hydrolysis-competent active site, ground state destabilization is unlikely to play a major role in enhancing the hydrolysis activity in myosin. Second, two-dimensional potential energy maps are used to better characterize the energetic landscape near the hydrolysis transition state. The results indicate that the transition state region is energetically flat and a range of structures representative of different mechanisms according to the classical nomenclature (e.g., “associative”, “dissociative” and “concerted”) are very close in energy. Therefore, at least in the case of ATP hydrolysis in myosin, the energetic distinction between different reaction mechanisms following the conventional nomenclature is likely small. This study highlights the importance of (i). explicitly evaluating the relevant energetic properties for determining whether a factor is essential

  16. Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

    SciTech Connect

    Martinson, E.A.; Goldstein, D.; Brown, J.H. )

    1989-09-05

    We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of (3H)choline and (3H)phosphorylcholine ((3H)Pchol) from cells containing (3H)choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of (3H)phosphatidic acid ((3H)PA) in cells containing (3H)myristate-labeled PC. (3H)Diacylglycerol ((3H)DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with (3H)myristate and (14C)arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol.

  17. [Plant signaling peptides. Cysteine-rich peptides].

    PubMed

    Ostrowski, Maciej; Kowalczyk, Stanisław

    2015-01-01

    Recent bioinformatic and genetic analyses of several model plant genomes have revealed the existence of a highly abundant group of signaling peptides that are defined as cysteine-rich peptides (CRPs). CRPs are usually in size between 50 and 90 amino acid residues, they are positively charged, and they contain 4-16 cysteine residues that are important for the correct conformational folding. Despite the structural differences among CRP classes, members from each class have striking similarities in their molecular properties and function. The present review presents the recent progress in research on signaling peptides from several families including: EPF/EPFL, SP11/SCR, PrsS, RALF, LURE, and some other peptides belonging to CRP group. There is convincing evidence indicating multiple roles for these CRPs as signaling molecules during the plant life cycle, ranging from stomata development and patterning, self-incompatibility, pollen tube growth and guidance, reproductive processes, and nodule formation. PMID:26281357

  18. Cell Penetrating Peptides and Cationic Antibacterial Peptides

    PubMed Central

    Rodriguez Plaza, Jonathan G.; Morales-Nava, Rosmarbel; Diener, Christian; Schreiber, Gabriele; Gonzalez, Zyanya D.; Lara Ortiz, Maria Teresa; Ortega Blake, Ivan; Pantoja, Omar; Volkmer, Rudolf; Klipp, Edda; Herrmann, Andreas; Del Rio, Gabriel

    2014-01-01

    Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs. PMID:24706763

  19. Effect of enzymatic hydrolysis of pancreatin and alcalase enzyme on some properties of edible bird's nest hydrolysate

    NASA Astrophysics Data System (ADS)

    Khushairay, Etty Syarmila Ibrahim; Ayub, Mohd Khan; Babji, Abdul Salam

    2014-09-01

    Edible bird nest (EBN) is a dried glutinous secretion from the salivary glands of several different swiftlet species. It is widely consumed as a health food due to its high beneficial effects to human health and has been considered to be one of the most precious food items by the Chinese for thousands of years. The aim of this study was to evaluate the effect of enzymatic hydrolysis on protein solubility (μg/g), the degree of hydrolysis (DH%), and peptide content (μg/g) of edible bird's nest hydrolysate. Initial protein solubility of boiled EBN was 25.5mg/g EBN. Treating the solubilized EBN with pancreatin 4NF for 1.0 - 1.5hours increased EBN protein solubility to 163.9mg/g and produced hydrolysate with DH% of 86.5% and 109.5mg/g peptide. EBN hydrolyzed with alcalase for 1.5 hours produced hydrolysate with protein solubility of 86.7mg/g, 82.7 DH% and had 104.1mg/g peptide content.

  20. Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

    PubMed Central

    Koch, Miriam; Flür, Sara; Kreutz, Christoph; Ennifar, Eric; Micura, Ronald; Polacek, Norbert

    2015-01-01

    Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases. PMID:25941362

  1. Adenosine triphosphate hydrolysis mechanism in kinesin studied by combined quantum-mechanical/molecular-mechanical metadynamics simulations.

    PubMed

    McGrath, Matthew J; Kuo, I-F Will; Hayashi, Shigehiko; Takada, Shoji

    2013-06-19

    Kinesin is a molecular motor that hydrolyzes adenosine triphosphate (ATP) and moves along microtubules against load. While motility and atomic structures have been well-characterized for various members of the kinesin family, not much is known about ATP hydrolysis inside the active site. Here, we study ATP hydrolysis mechanisms in the kinesin-5 protein Eg5 by using combined quantum mechanics/molecular mechanics metadynamics simulations. Approximately 200 atoms at the catalytic site are treated by a dispersion-corrected density functional and, in total, 13 metadynamics simulations are performed with their cumulative time reaching ~0.7 ns. Using the converged runs, we compute free energy surfaces and obtain a few hydrolysis pathways. The pathway with the lowest free energy barrier involves a two-water chain and is initiated by the Pγ-Oβ dissociation concerted with approach of the lytic water to PγO3-. This immediately induces a proton transfer from the lytic water to another water, which then gives a proton to the conserved Glu270. Later, the proton is transferred back from Glu270 to HPO(4)2- via another hydrogen-bonded chain. We find that the reaction is favorable when the salt bridge between Glu270 in switch II and Arg234 in switch I is transiently broken, which facilitates the ability of Glu270 to accept a proton. When ATP is placed in the ADP-bound conformation of Eg5, the ATP-Mg moiety is surrounded by many water molecules and Thr107 blocks the water chain, which together make the hydrolysis reaction less favorable. The observed two-water chain mechanisms are rather similar to those suggested in two other motors, myosin and F1-ATPase, raising the possibility of a common mechanism. PMID:23751065

  2. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions. PMID:26374891

  3. Coarse Graining to Investigate Membrane Induced Peptide Folding of Anticancer Peptides

    NASA Astrophysics Data System (ADS)

    Ganesan, Sai; Xu, Hongcheng; Matysiak, Silvina

    Information about membrane induced peptide folding mechanisms using all-atom molecular dynamics simulations is a challenge due to time and length scale issues.We recently developed a low resolution Water Explicit Polarizable PROtein coarse-grained Model by adding oppositely charged dummy particles inside protein backbone beads.These two dummy particles represent a fluctuating dipole,thus introducing structural polarization into the coarse-grained model.With this model,we were able to achieve significant α- β secondary structure content de novo,without any added bias.We extended the model to zwitterionic and anionic lipids,by adding oppositely charged dummy particles inside polar beads, to capture the ability of the head group region to form hydrogen bonds.We use zwitterionic POPC and anionic POPS as our model lipids, and a cationic anticancer peptide,SVS1,as our model peptide.We have characterized the driving forces for SVS1 folding on lipid bilayers with varying anionic and zwitterionic lipid compositions.Based on our results, dipolar interactions between peptide backbone and lipid head groups contribute to stabilize folded conformations.Cooperativity in folding is induced by both intra peptide and membrane-peptide interaction.

  4. Peptide Nanovesicles Formed by the Self-Assembly of Branched Amphiphilic Peptides

    PubMed Central

    Gudlur, Sushanth; Sukthankar, Pinakin; Gao, Jian; Avila, L. Adriana; Hiromasa, Yasuaki; Chen, Jianhan; Iwamoto, Takeo; Tomich, John M.

    2012-01-01

    Peptide-based packaging systems show great potential as safer drug delivery systems. They overcome problems associated with lipid-based or viral delivery systems, vis-a-vis stability, specificity, inflammation, antigenicity, and tune-ability. Here, we describe a set of 15 & 23-residue branched, amphiphilic peptides that mimic phosphoglycerides in molecular architecture. These peptides undergo supramolecular self-assembly and form solvent-filled, bilayer delimited spheres with 50–200 nm diameters as confirmed by TEM, STEM and DLS. Whereas weak hydrophobic forces drive and sustain lipid bilayer assemblies, these all-peptide structures are stabilized potentially by both hydrophobic interactions and hydrogen bonds and remain intact at low micromolar concentrations and higher temperatures. A linear peptide lacking the branch point showed no self-assembly properties. We have observed that these peptide vesicles can trap fluorescent dye molecules within their interior and are taken up by N/N 1003A rabbit lens epithelial cells grown in culture. These assemblies are thus potential drug delivery systems that can overcome some of the key limitations of the current packaging systems. PMID:23028970

  5. A novel reaction mediated by human aldehyde oxidase: amide hydrolysis of GDC-0834.

    PubMed

    Sodhi, Jasleen K; Wong, Susan; Kirkpatrick, Donald S; Liu, Lichuan; Khojasteh, S Cyrus; Hop, Cornelis E C A; Barr, John T; Jones, Jeffrey P; Halladay, Jason S

    2015-06-01

    GDC-0834, a Bruton's tyrosine kinase inhibitor investigated as a potential treatment of rheumatoid arthritis, was previously reported to be extensively metabolized by amide hydrolysis such that no measurable levels of this compound were detected in human circulation after oral administration. In vitro studies in human liver cytosol determined that GDC-0834 (R)-N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo- 4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b] thiophene-2-carboxamide) was rapidly hydrolyzed with a CLint of 0.511 ml/min per milligram of protein. Aldehyde oxidase (AO) and carboxylesterase (CES) were putatively identified as the enzymes responsible after cytosolic fractionation and mass spectrometry-proteomics analysis of the enzymatically active fractions. Results were confirmed by a series of kinetic experiments with inhibitors of AO, CES, and xanthine oxidase (XO), which implicated AO and CES, but not XO, as mediating GDC-0834 amide hydrolysis. Further supporting the interaction between GDC-0834 and AO, GDC-0834 was shown to be a potent reversible inhibitor of six known AO substrates with IC50 values ranging from 0.86 to 1.87 μM. Additionally, in silico modeling studies suggest that GDC-0834 is capable of binding in the active site of AO with the amide bond of GDC-0834 near the molybdenum cofactor (MoCo), orientated in such a way to enable potential nucleophilic attack on the carbonyl of the amide bond by the hydroxyl of MoCo. Together, the in vitro and in silico results suggest the involvement of AO in the amide hydrolysis of GDC-0834. PMID:25845827

  6. Limitations in Bonding to Dentin and Experimental Strategies to Prevent Bond Degradation

    PubMed Central

    Liu, Y.; Tjäderhane, L.; Breschi, L.; Mazzoni, A.; Li, N.; Mao, J.; Pashley, D.H.; Tay, F.R.

    2011-01-01

    The limited durability of resin-dentin bonds severely compromises the lifetime of tooth-colored restorations. Bond degradation occurs via hydrolysis of suboptimally polymerized hydrophilic resin components and degradation of water-rich, resin-sparse collagen matrices by matrix metalloproteinases (MMPs) and cysteine cathepsins. This review examined data generated over the past three years on five experimental strategies developed by different research groups for extending the longevity of resin-dentin bonds. They include: (1) increasing the degree of conversion and esterase resistance of hydrophilic adhesives; (2) the use of broad-spectrum inhibitors of collagenolytic enzymes, including novel inhibitor functional groups grafted to methacrylate resins monomers to produce anti-MMP adhesives; (3) the use of cross-linking agents for silencing the activities of MMP and cathepsins that irreversibly alter the 3-D structures of their catalytic/allosteric domains; (4) ethanol wet-bonding with hydrophobic resins to completely replace water from the extrafibrillar and intrafibrillar collagen compartments and immobilize the collagenolytic enzymes; and (5) biomimetic remineralization of the water-filled collagen matrix using analogs of matrix proteins to progressively replace water with intrafibrillar and extrafibrillar apatites to exclude exogenous collagenolytic enzymes and fossilize endogenous collagenolytic enzymes. A combination of several of these strategies should result in overcoming the critical barriers to progress currently encountered in dentin bonding. PMID:21220360

  7. Surface-Bonded Antimicrobial Activity of an Organosilicon Quaternary Ammonium Chloride

    PubMed Central

    Isquith, A. J.; Abbott, E. A.; Walters, P. A.

    1972-01-01

    The hydrolysis product of 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride exhibited antimicrobial activity against a broad range of microorganisms while chemically bonded to a variety of surfaces. The chemical was not removed from surfaces by repeated washing with water, and its antimicrobial activity could not be attributed to a slow release of the chemical, but rather to the surface-bonded chemical. Images PMID:4650597

  8. Hydrolysis of DFP and the Nerve Agent (S)-Sarin by DFPase Proceeds Along Two Different Reaction Pathways: Implica-tions for Engineering Bioscavengers

    SciTech Connect

    Wymore, Troy W; Langan, Paul; Smith, Jeremy C; Field, Martin J.; Parks, Jerry M

    2014-01-01

    Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents ( bioscavengers ) is an emerging prophylactic approach to diminishing their toxic effects. Although its native function is not known, diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical (QM/MM) umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca2+, donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily due to the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed -propeller folds for more efficient degradation of OP compounds.

  9. Hydrolysis of DFP and the nerve agent (S)-sarin by DFPase proceeds along two different reaction pathways: implications for engineering bioscavengers.

    PubMed

    Wymore, Troy; Field, Martin J; Langan, Paul; Smith, Jeremy C; Parks, Jerry M

    2014-05-01

    Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents ("bioscavengers") is an emerging prophylactic approach to diminish their toxic effects. Although its native function is not known, diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P-F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca(2+), donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily because of the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed β-propeller folds for more efficient degradation of OP compounds. PMID:24720808

  10. Hydrolysis of DFP and the Nerve Agent (S)-Sarin by DFPase Proceeds along Two Different Reaction Pathways: Implications for Engineering Bioscavengers

    PubMed Central

    2015-01-01

    Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents (“bioscavengers”) is an emerging prophylactic approach to diminish their toxic effects. Although its native function is not known, diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P–F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca2+, donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily because of the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed β-propeller folds for more efficient degradation of OP compounds. PMID:24720808

  11. Diffusion bonding aeroengine components

    NASA Astrophysics Data System (ADS)

    Fitzpatrick, G. A.; Broughton, T.

    1988-10-01

    The use of diffusion bonding processes at Rolls-Royce for the manufacture of titanium-alloy aircraft engine components and structures is described. A liquid-phase diffusion bonding process called activated diffusion bonding has been developed for the manufacture of the hollow titanium wide chord fan blade. In addition, solid-state diffusion bonding is being used in the manufacture of hollow vane/blade airfoil constructions mainly in conjunction with superplastic forming and hot forming techniques.

  12. Rapid adhesive bonding concepts

    NASA Technical Reports Server (NTRS)

    Stein, B. A.; Tyeryar, J. R.; Hodges, W. T.

    1984-01-01

    Adhesive bonding in the aerospace industry typically utilizes autoclaves or presses which have considerable thermal mass. As a consequence, the rates of heatup and cooldown of the bonded parts are limited and the total time and cost of the bonding process is often relatively high. Many of the adhesives themselves do not inherently require long processing times. Bonding could be performed rapidly if the heat was concentrated in the bond lines or at least in the adherends. Rapid adhesive bonding concepts were developed to utilize induction heating techniques to provide heat directly to the bond line and/or adherends without heating the entire structure, supports, and fixtures of a bonding assembly. Bonding times for specimens are cut by a factor of 10 to 100 compared to standard press bonding. The development of rapid adhesive bonding for lap shear specimens (per ASTM D1003 and D3163), for aerospace panel bonding, and for field repair needs of metallic and advanced fiber reinforced polymeric matrix composite structures are reviewed.

  13. β-Turn Sequences Promote Stability of Peptide Substrates for Kinases within the Cytosolic Environment

    PubMed Central

    Yang, Shan; Proctor, Angela; Cline, Lauren L.; Houston, Kaiulani M.; Waters, Marcey L.; Allbritton, Nancy L.

    2013-01-01

    A strategy was developed to extend the lifetime of an peptide-based substrate for Abl kinase in the cytosolic environment. Small β-turn structures were added to the peptide’s N-terminus to block entry into peptidase catalytic sites. The influence of the size of the β-turn and two covalent cross-linking strategies on the rate of hydrolysis was assessed. The most peptidase-resistant substrate was degraded at a rate of 0.6 pmol mg−1 s−1 and possessed a half-life of 20.3 ± 1.7 min in a Baf/BCR-ABL cytosolic lysate, representing 16- and 40-fold improvements, respectively, over that of a control peptide lacking the β-turn structure. Furthermore, the kcat/KM value of this peptide was 432 μM−1 min−1, a 1.25X increase over the unmodified control, verifying that the added β-turn did not hinder the substrate properties of the peptide. This improved peptide was microinjected into single Baf/BCR-ABL cells and substrate phosphorylation measured. Zero to forty percent of the peptide was phosphorylated in the single cells. In contrast, when the control peptide without a β-turn was loaded into cells, the peptide was too rapidly degraded to detect phosphorylation. This work demonstrates that small β-turn structures can render peptides more resistant to hydrolysis while retaining substrate efficacy and shows that these stabilized peptides have the potential to be of high utility in single-cell enzyme assays. PMID:23785707

  14. Rational design of a series of novel amphipathic cell-penetrating peptides.

    PubMed

    Regberg, Jakob; Srimanee, Artita; Erlandsson, Mikael; Sillard, Rannar; Dobchev, Dimitar A; Karelson, Mati; Langel, Ulo

    2014-04-10

    A series of novel, amphipathic cell-penetrating peptides was developed based on a combination of the model amphipathic peptide sequence and modifications based on the strategies developed for PepFect and NickFect peptides. The aim was to study the role of amphipathicity for peptide uptake and to investigate if the modifications developed for PepFect peptides could be used to improve the uptake of another class of cell-penetrating peptides. The peptides were synthesized by solid phase peptide synthesis and characterized by circular dichroism spectroscopy. Non-covalent peptide-plasmid complexes were formed by co-incubation of the peptides and plasmids in water solution. The complexes were characterized by dynamic light scattering and cellular uptake of the complexes was studied in a luciferase-based plasmid transfection assay. A quantitative structure-activity relationship (QSAR) model of cellular uptake was developed using descriptors including hydrogen bonding, peptide charge and positions of nitrogen atoms. The peptides were found to be non-toxic and could efficiently transfect cells with plasmid DNA. Cellular uptake data was correlated to QSAR predictions and the predicted biological effects obtained from the model correlated well with experimental data. The QSAR model could improve the understanding of structural requirements for cell penetration, or could potentially be used to predict more efficient cell-penetrating peptides. PMID:24463071

  15. Enzymatic hydrolysis of PTT polymers and oligomers.

    PubMed

    Eberl, A; Heumann, S; Kotek, R; Kaufmann, F; Mitsche, S; Cavaco-Paulo, A; Gübitz, G M

    2008-05-20

    Oligomers and polymers (film, fabrics) of the linear aromatic polyester poly(trimethylene terephthalate) (PTT) were treated with polyesterases from Thermomyces lanuginosus, Penicillium citrinum, Thermobifida fusca and Fusarium solani pisi. The cutinase from T. fusca was found to release the highest amounts of hydrolysis products from PTT materials and was able to open and hydrolyse a cyclic PTT dimer according to RP-HPLC-UV detection. In contrast, the lipase from T. lanuginosus also showed activity on the PTT fibres and on bis(3-hydroxypropyl) terephthalate (BHPT) but was not able to hydrolyse the polymer film, mono(3-hydroxypropyl) terephthalate (MHPT) nor the cyclic dimer of PTT. As control enzymes inhibited with mercury chloride were used. Surface hydrophilicity changes were investigated with contact angle measurements and the degree of crystallinity changes were determined with DSC. PMID:18405994

  16. Catalytic Zinc Complexes for Phosphate Diester Hydrolysis**

    PubMed Central

    Tirel, Emmanuel Y; Bellamy, Zoë; Adams, Harry; Lebrun, Vincent; Duarte, Fernanda; Williams, Nicholas H

    2014-01-01

    Creating efficient artificial catalysts that can compete with biocatalysis has been an enduring challenge which has yet to be met. Reported herein is the synthesis and characterization of a series of zinc complexes designed to catalyze the hydrolysis of phosphate diesters. By introducing a hydrated aldehyde into the ligand we achieve turnover for DNA-like substrates which, combined with ligand methylation, increases reactivity by two orders of magnitude. In contrast to current orthodoxy and mechanistic explanations, we propose a mechanism where the nucleophile is not coordinated to the metal ion, but involves a tautomer with a more effective Lewis acid and more reactive nucleophile. This data suggests a new strategy for creating more efficient metal ion based catalysts, and highlights a possible mode of action for metalloenzymes. PMID:24919567

  17. Pretreatment and enzymatic hydrolysis of corn fiber

    SciTech Connect

    Grohmann, K.; Bothast, R.J.

    1996-10-01

    Corn fiber is a co-product of the corn wet milling industry which is usually marketed as a low value animal feed ingredient. Approximately 1.2 x 10{sup 6} dry tons of this material are produced annually in the United States. The fiber is composed of kernel cell wall fractions and a residual starch which can all be potentially hydrolyzed to a mixture of glucose, xylose, arabinose and galactose. We have investigated a sequential saccharification of polysaccharides in corn fiber by a treatment with dilute sulfuric acid at 100 to 160{degrees}C followed by partial neutralization and enzymatic hydrolysis with mixed cellulose and amyloglucosidase enzymes at 45{degrees}C. The sequential treatment achieved a high (approximately 85%) conversion of all polysaccharides in the corn fiber to monomeric sugars, which were in most cases fermentable to ethanol by the recombinant bacterium Escherichia coli KOll.

  18. Fermentable sugars by chemical hydrolysis of biomass

    PubMed Central

    Binder, Joseph B.; Raines, Ronald T.

    2010-01-01

    Abundant plant biomass has the potential to become a sustainable source of fuels and chemicals. Realizing this potential requires the economical conversion of recalcitrant lignocellulose into useful intermediates, such as sugars. We report a high-yielding chemical process for the hydrolysis of biomass into monosaccharides. Adding water gradually to a chloride ionic liquid-containing catalytic acid leads to a nearly 90% yield of glucose from cellulose and 70–80% yield of sugars from untreated corn stover. Ion-exclusion chromatography allows recovery of the ionic liquid and delivers sugar feedstocks that support the vigorous growth of ethanologenic microbes. This simple chemical process, which requires neither an edible plant nor a cellulase, could enable crude biomass to be the sole source of carbon for a scalable biorefinery. PMID:20194793

  19. Light-Switchable Peptides with a Hemithioindigo Unit: Peptide Design, Photochromism, and Optical Spectroscopy.

    PubMed

    Kitzig, S; Thilemann, M; Cordes, T; Rück-Braun, Karola

    2016-05-01

    This Minireview focuses on the hemithioindigo photoswitch and its use for the reversible control of three-dimensional peptide structure and related biological functions. Both the general design aspects and biophysical properties of various hemithioindigo-based chromopeptides are summarized. Hemithioindigo undergoes reversible Z→E photoisomerization after absorption of visible light. The unique ultrafast switching mechanism of hemithioindigo combines picosecond isomerization kinetics with strong double-bond torsion after light absorption, making it the ideal tool for instantaneous modulation of biological structure. Various inhibitors and model peptides based on hemithioindigo are described that can directly regulate biological signaling or allow the fastest events in peptide folding to be studied. Finally, a diverse range of chromopeptides with photoswitchable β-hairpin structures based on azobenzenes, stilbenes, and hemithioindigo are compared to emphasize the unique properties of hemithioindigo. PMID:26789782

  20. Antihypertensive peptides from curd

    PubMed Central

    Dabarera, Melani Chathurika; Athiththan, Lohini V.; Perera, Rasika P.

    2015-01-01

    Introduction: Curd (Dadhi) peptides reduce hypertension by inhibiting angiotensin converting enzyme (ACE) and serum cholesterol. Peptides vary with bacterial species and milk type used during fermentation. Aim: To isolate and assay the antihypertensive peptides, before and after digestion, in two commercially available curd brands in Sri Lanka. Materials and Methods: Whey (Dadhi Mastu) separated by high-speed centrifugation was isolated using reverse-phase-high- performance liquid chromatography (HPLC). Eluted fractions were analyzed for ACE inhibitory activity using modified Cushman and Cheung method. Curd samples were subjected to enzymatic digestion with pepsin, trypsin, and carboxypeptidase-A at their optimum pH and temperature. Peptides isolated using reverse-phase-HPLC was assayed for ACE inhibitory activity. Results: Whey peptides of both brands gave similar patterns (seven major and five minor peaks) in HPLC elution profile. Smaller peptides concentration was higher in brand 1 and penta-octapeptides in brand 2. Pentapeptide had the highest ACE inhibitory activity (brand 2–90% and brand 1–73%). After digestion, di and tri peptides with similar inhibitory patterns were obtained in both which were higher than before digestion. Thirteen fractions were obtained, where nine fractions showed more than 70% inhibition in both brands with 96% ACE inhibition for a di-peptide. Conclusion: Curd has ACE inhibitory peptides and activity increases after digestion. PMID:27011726

  1. Antimicrobial Peptides in Reptiles

    PubMed Central

    van Hoek, Monique L.

    2014-01-01

    Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

  2. Polycyclic peptide therapeutics.

    PubMed

    Baeriswyl, Vanessa; Heinis, Christian

    2013-03-01

    Owing to their excellent binding properties, high stability, and low off-target toxicity, polycyclic peptides are an attractive molecule format for the development of therapeutics. Currently, only a handful of polycyclic peptides are used in the clinic; examples include the antibiotic vancomycin, the anticancer drugs actinomycin D and romidepsin, and the analgesic agent ziconotide. All clinically used polycyclic peptide drugs are derived from natural sources, such as soil bacteria in the case of vancomycin, actinomycin D and romidepsin, or the venom of a fish-hunting coil snail in the case of ziconotide. Unfortunately, nature provides peptide macrocyclic ligands for only a small fraction of therapeutic targets. For the generation of ligands of targets of choice, researchers have inserted artificial binding sites into natural polycyclic peptide scaffolds, such as cystine knot proteins, using rational design or directed evolution approaches. More recently, large combinatorial libraries of genetically encoded bicyclic peptides have been generated de novo and screened by phage display. In this Minireview, the properties of existing polycyclic peptide drugs are discussed and related to their interesting molecular architectures. Furthermore, technologies that allow the development of unnatural polycyclic peptide ligands are discussed. Recent application of these technologies has generated promising results, suggesting that polycyclic peptide therapeutics could potentially be developed for a broad range of diseases. PMID:23355488

  3. Peptide folding simulations.

    PubMed

    Gnanakaran, S; Nymeyer, Hugh; Portman, John; Sanbonmatsu, Kevin Y; García, Angel E

    2003-04-01

    Developments in the design of small peptides that mimic proteins in complexity, recent advances in nanosecond time-resolved spectroscopy methods to study peptides and the development of modern, highly parallel simulation algorithms have come together to give us a detailed picture of peptide folding dynamics. Two newly implemented simulation techniques, parallel replica dynamics and replica exchange molecular dynamics, can now describe directly from simulations the kinetics and thermodynamics of peptide formation, respectively. Given these developments, the simulation community now has the tools to verify and validate simulation protocols and models (forcefields). PMID:12727509

  4. Exploration of the mechanism of the hydrolysis of chlorine nitrate in small water clusters using electronic structure methods

    SciTech Connect

    McNamara, J.P.; Hillier, I.H.

    1999-09-09

    High-level electronic structure calculations have been used to study the mechanism of hydrolysis of chlorine nitrate in neutral water clusters containing three to eight solvating water molecules. The calculations clarify some of the current uncertainties in the hydrolysis mechanism. As the size of the water cluster is increased, ClONO{sub 2} shows increasing ionization along the O{sub 2}NO-Cl bond consistent with the proposed predissociation in which the electrophilicity of the chlorine atom is enhanced, thus making it more susceptible to nucleophilic attack from a surface water molecule. A species akin to the experimentally observed intermediate, H{sub 2}OCl{sup +} is found to be stable in a cluster containing eight water molecules. The hydrolysis products, ionized nitric (H{sub 3}O{sup +}/NO{sub 3}{sup {minus}}) and molecular hypochlorous (HOCl) acids, are found to be stable in two different types of structures, containing six and eight water molecules. For the water cluster containing six water molecules, which has a structure related to ordinary hexagonal ice, ClONO{sub 2} is hydrolyzed to yield H{sub 3}O{sup +}/NO{sub 3}{sup {minus}}/HOCl, with essentially no barrier. The calculations thus predict that hydrolysis of ClONO{sub 2} on PSC ice aerosols can proceed spontaneously in small neutral water clusters.

  5. Does Electron Capture Dissociation Cleave Protein Disulfide Bonds?

    PubMed Central

    Ganisl, Barbara; Breuker, Kathrin

    2012-01-01

    Peptide and protein characterization by mass spectrometry (MS) relies on their dissociation in the gas phase into specific fragments whose mass values can be aligned as ‘mass ladders’ to provide sequence information and to localize possible posttranslational modifications. The most common dissociation method involves slow heating of even-electron (M+n H)n+ ions from electrospray ionization by energetic collisions with inert gas, and cleavage of amide backbone bonds. More recently, dissociation methods based on electron capture or transfer were found to provide far more extensive sequence coverage through unselective cleavage of backbone N–Cα bonds. As another important feature of electron capture dissociation (ECD) and electron transfer dissociation (ETD), their unique unimolecular radical ion chemistry generally preserves labile posttranslational modifications such as glycosylation and phosphorylation. Moreover, it was postulated that disulfide bond cleavage is preferred over backbone cleavage, and that capture of a single electron can break both a backbone and a disulfide bond, or even two disulfide bonds between two peptide chains. However, the proposal of preferential disulfide bond cleavage in ECD or ETD has recently been debated. The experimental data presented here reveal that the mechanism of protein disulfide bond cleavage is much more intricate than previously anticipated. PMID:24363980

  6. Electron Transfer Dissociation Reveals Changes in the Cleavage Frequencies of Backbone Bonds Distant to Amide-to-Ester Substitutions in Polypeptides

    NASA Astrophysics Data System (ADS)

    Hansen, Thomas A.; Jung, Hye R.; Kjeldsen, Frank

    2011-11-01

    Interrogation of electron transfer dissociation (ETD) mass spectra of peptide amide-to-ester backbone bond substituted analogues (depsipeptides) reveals substantial differences in the entire backbone cleavage frequencies. It is suggested that the point permutation of backbone bonds leads to changes in the predominant ion structures by removal/weakening of specific hydrogen bonding. ETD responds to these changes by redistributing the cleavage frequencies of the peptide backbone bonds. In comparison, no distinction between depsi-/peptide was observed using collision-activated dissociation, which is consistent with a general unfolding and elimination of structural information of these ions. These results should encourage further exploration of depsipeptides for gas-phase structural characterization.

  7. Bonding thermoplastic polymers

    DOEpatents

    Wallow, Thomas I.; Hunter, Marion C.; Krafcik, Karen Lee; Morales, Alfredo M.; Simmons, Blake A.; Domeier, Linda A.

    2008-06-24

    We demonstrate a new method for joining patterned thermoplastic parts into layered structures. The method takes advantage of case-II permeant diffusion to generate dimensionally controlled, activated bonding layers at the surfaces being joined. It is capable of producing bonds characterized by cohesive failure while preserving the fidelity of patterned features in the bonding surfaces. This approach is uniquely suited to production of microfluidic multilayer structures, as it allows the bond-forming interface between plastic parts to be precisely manipulated at micrometer length scales. The bond enhancing procedure is easily integrated in standard process flows and requires no specialized equipment.

  8. Prospective bonding applications

    NASA Astrophysics Data System (ADS)

    Ancenay, H.; Benazet, D.

    1981-07-01

    Adhesive bonding in industry and in the laboratory is surveyed and prospects for its wider utilization are assessed. The economic impact of bonding technology on industry is discussed. Research is reviewed, centering on the development of nondestructive testing and inspection techniques. Traditional (wood) as well as new materials susceptible to bonding are considered. Applications in construction and civil engineering, in aeronautics, and in the automobile industry are covered. The use of glues in mechanical constructions, in assembling cylindrical parts, and in metal-metal bonding are examined. Hybrid assembling and bonding of composite materials are included.

  9. Bioabatement with hemicellulase supplementation to reduce enzymatic hydrolysis inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Removal of inhibitory compounds by bioabatement, combined with xylan hydrolysis, enables effective cellulose hydrolysis of pretreated corn stover, for fermentation of the sugars to fuel ethanol or other products. The fungus Coniochaeta ligniaria NRRL30616 eliminates most enzyme and fermentation inhi...

  10. Ultrasound Enhancement of Enzymatic Hydrolysis of Cellulose Plant Matter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The work reported here is based on acceleration of enzymatic hydrolysis of plant biomass substrate by introduction of low intensity, uniform ultrasound field into a reaction chamber (bio-reactor). This method may serve as improvement of rates in the hydrolysis of cellulosic materials to sugars, whi...

  11. Class Projects in Physical Organic Chemistry: The Hydrolysis of Aspirin

    ERIC Educational Resources Information Center

    Marrs, Peter S.

    2004-01-01

    An exercise that provides a hands-on demonstration of the hydrolysis of aspirin is presented. The key to understanding the hydrolysis is recognizing that all six process may occur simultaneously and that the observed rate constant is the sum of the rate constants that one rate constant dominates the overall process.

  12. Enhanced functional properties of tannic acid after thermal hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thermal hydrolysis processing of fresh tannic acid was carried out in a closed reactor at four different temperatures (65, 100, 150 and 200°C). Pressures reached in the system were 1.3 and 4.8 MPa at 150 and 200°C, respectively. Hydrolysis products (gallic acid and pyrogallol) were separated and qua...

  13. Peptide Conjugation to a Polymer Coating via Native Chemical Ligation of Azlactones for Cell Culture.

    PubMed

    Schmitt, Samantha K; Trebatoski, David J; Krutty, John D; Xie, Angela W; Rollins, Benjamin; Murphy, William L; Gopalan, Padma

    2016-03-14

    Conjugation of biomolecules for stable presentation is an essential step toward reliable chemically defined platforms for cell culture studies. In this work, we describe the formation of a stable and site-specific amide bond via the coupling of a cysteine terminated peptide at low concentration to an azlactone containing copolymer coating. A copolymer of polyethylene glycol methyl ether methacrylate-ran-vinyl azlactone-ran-glycidyl methacrylate P(PEGMEMA-r-VDM-r-GMA) was used to form a thin coating (20-30 nm) on silicon and polycarbonate substrates. The formation and stability of coating-peptide bonds for peptides containing free thiols and amines were quantified by X-ray photoelectron spectroscopy (XPS) after exposure to cell culture conditions. Peptides containing a thiol as the only nucleophile coupled via a thioester bond; however, the bond was labile under cell culture conditions and almost all the bound peptides were displaced from the surface over a period of 2 days. Coupling with N-terminal primary amine peptides resulted in the formation of an amide bond with low efficiency (<20%). In contrast, peptides containing an N-terminal cysteine, which contain both nucleophiles (free thiol and amine) in close proximity, bound with 67% efficiency under neutral pH, and were stable under the same conditions for 2 weeks. Control studies confirm that the stable amide formation was a result of an intramolecular rearrangement through a N-acyl intermediate that resembles native chemical ligation. Through a combination of XPS and cell culture studies, we show that the cysteine terminated peptides undergo a native chemical ligation process at low peptide concentration in aqueous media, short reaction time, and at room temperature resulting in the stable presentation of peptides beyond 2 weeks for cell culture studies. PMID:26835552

  14. Simple Bond Cleavage

    SciTech Connect

    Gary S. Groenewold

    2005-08-01

    Simple bond cleavage is a class of fragmentation reactions in which a single bond is broken, without formation of new bonds between previously unconnected atoms. Because no bond making is involved, simple bond cleavages are endothermic, and activation energies are generally higher than for rearrangement eliminations. The rate of simple bond cleavage reactions is a strong function of the internal energy of the molecular ion, which reflects a loose transition state that resembles reaction products, and has a high density of accessible states. For this reason, simple bond cleavages tend to dominate fragmentation reactions for highly energized molecular ions. Simple bond cleavages have negligible reverse activation energy, and hence they are used as valuable probes of ion thermochemistry, since the energy dependence of the reactions can be related to the bond energy. In organic mass spectrometry, simple bond cleavages of odd electron ions can be either homolytic or heterolytic, depending on whether the fragmentation is driven by the radical site or the charge site. Simple bond cleavages of even electron ions tend to be heterolytic, producing even electron product ions and neutrals.

  15. Amine-Promoted Organosilicate Hydrolysis Mechanism at Near-Neutral pH

    NASA Astrophysics Data System (ADS)

    Delak, K. M.; Sahai, N.

    2006-12-01

    Proteins bearing polylysine moeities and histidine and serine amino-aicd residues, isolated from diatoms and sponges, are known to promote biological nanoporous silica formation [1, 2]. Using 29Si NMR, we have shown quantitatively that monoamines and small polyamines can chemically accelerate the hydrolysis and condensation rates of organosilicate starting materials, in biomimetic silica synthesis pathways, at circum- neutral pHs and room temperature [3, 4]. The present study is focused on understanding the mechanistic role of these amines in catalyzing the hydrolysis step that precedes condensation [5]. We conducted 29Si NMR experimental studies over a range of temperature and pHs for the hydrolysis rates of trimethylethoxysilane (TMES), a model compound with only one hydrolyzable bond. Experimental results were combined with quantum mechanical hybrid Density Functional Theory calculations of putative intermediate and transition state structures for TMES and tetramethylorthosilicate (TMOS) which has four hydrolyzable bonds. Comparison of calculated energies with experimentally-determined activation energies indicated that amines promote TMES hydrolysis mainly due to the amine's acidity at neutral pH. The proton released by the amine is transferred to the organosilicate, producing a protonated, ethoxy leaving group that can be displaced by water in an SN2 reaction. For TMOS, the activation energy of proton-transfer coupled with SN2 substitution is comparable to that for Corriu's nucleophile-activated nucleophilic displacement mechanism [6], such that the mechanism of amine-catalyzed hydrolysis is mostly dependent on the ambient pH conditions as well as the type of amine. The molecular mechanisms of hydrolysis and aggregation are reflected, ultimately, on the larger scale in the silica morphology where amines promoting faster hydrolysis result in glassy products compared to slower hydrolyzing amines forming particulate silica [7, 8]. REFERENCES [1] Kroger N

  16. Insights into the molecular architecture of a peptide nanotube using FTIR and solid-state NMR spectroscopic measurements on an aligned sample.

    PubMed

    Middleton, David A; Madine, Jillian; Castelletto, Valeria; Hamley, Ian W

    2013-09-27

    Queuing up: Molecular orientation within macroscopically aligned nanotubes of the peptide AAAAAAK can be studied by solid-state NMR and IR spectroscopy. Line shape analysis of the NMR spectra indicates that the peptide N-H bonds are tilted 65-70° relative to the nanotube long axis. Re-evaluation of earlier X-ray fiber diffraction data suggests that the peptide molecules are hydrogen-bonded in a helical arrangement along the nanotube axis. PMID:23955926

  17. Dipeptidyl Peptidase IV-Inhibitory Peptides Derived from Silver Carp (Hypophthalmichthys molitrix Val.) Proteins.

    PubMed

    Zhang, Ying; Chen, Ran; Chen, Xiling; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

    2016-02-01

    The dipeptidyl peptidase IV (DPP-IV)-inhibitory bioactivity of silver carp protein (SCP) hydrolysates were investigated, and their containing efficacious DPP-IV-inhibitory peptides were explored by in silico hydrolysis analysis, peptide separation combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, and chemical synthesis. SCP hydrolysates generated by six proteases all showed efficient DPP-IV-inhibitory activities, and Neutrase-generated hydrolysates had the greatest DPP-IV inhibition (IC50 of 1.12 mg/mL). In silico Neutrase hydrolysis revealed hundreds of fragments released from myosin, actin, and collagen of SCPs, which include different Pro-motif peptides but only three reported peptidic DPP-IV inhibitors with moderate or weak bioactivity. In addition, three new DPP-IV-inhibitory peptides were identified using LC-MS/MS; in particular, LPIIDI and APGPAGP showed high DPP-IV-inhibitory activity with IC50 of 105.44 and 229.14 μM, respectively, and behaved in competitive/non-competitive mixed-type DPP-IV inhibition mode. The results indicate that the SCP-derived DPP-IV-inhibitory peptides could be potential functional ingredients in the diabetic diet. PMID:26758401

  18. Purification of a novel nitric oxide inhibitory peptide derived from enzymatic hydrolysates of Mytilus coruscus.

    PubMed

    Kim, Eun-Kyung; Kim, Yon-Suk; Hwang, Jin-Woo; Kang, Seo Hee; Choi, Dong-Kug; Lee, Kwang-Ho; Lee, Jung Suck; Moon, Sang-Ho; Jeon, Byong-Tae; Park, Pyo-Jam

    2013-06-01

    Shellfish contain significant levels of high quality protein and are therefore a potential source for biofunctional high-value peptides. To purify a novel anti-inflammatory peptide from Mytilus coruscus (M. coruscus), we applied enzymatic hydrolysis and tangential flow filtration (TFF) and investigated its nitric oxide inhibitory property. To prepare the peptide, eight proteases were employed for enzymatic hydrolysis. Flavouzyme hydrolysates, which showed clearly superior nitric oxide inhibitory activity on lipopolysaccharide (LPS)-stimulated RAW264.7, were further purified using a TFF system and consecutive chromatographic methods. Finally, a novel anti-inflammatory peptide composed of 10 amino acid residues was obtained, and the sequence was identified as Gly-Val-Ser-Leu-Leu-Gln-Gln-Phe-Phe-Leu at N-terminal position. The peptide from M. coruscus effectively inhibited nitric oxide production on macrophage cells. This is the first report of an anti-inflammatory peptide derived from the hydrolysates of M. coruscus. PMID:23500953

  19. Trihalomethane hydrolysis in drinking water at elevated temperatures.

    PubMed

    Zhang, Xiao-Lu; Yang, Hong-Wei; Wang, Xiao-Mao; Karanfil, Tanju; Xie, Yuefeng F

    2015-07-01

    Hydrolysis could contribute to the loss of trihalomethanes (THMs) in the drinking water at elevated temperatures. This study was aimed at investigating THM hydrolysis pertaining to the storage of hot boiled water in enclosed containers. The water pH value was in the range of 6.1-8.2 and the water temperature was varied from 65 to 95 °C. The effects of halide ions, natural organic matter, and drinking water matrix were investigated. Results showed that the hydrolysis rates declined in the order following CHBrCl2 > CHBr2Cl > CHBr3 > CHCl3. THM hydrolysis was primarily through the alkaline pathway, except for CHCl3 in water at relatively low pH value. The activation energies for the alkaline hydrolysis of CHCl3, CHBrCl2, CHBr2Cl and CHBr3 were 109, 113, 115 and 116 kJ/mol, respectively. No hydrolysis intermediates could accumulate in the water. The natural organic matter, and probably other constituents, in drinking water could substantially decrease THM hydrolysis rates by more than 50%. When a drinking water was at 90 °C or above, the first order rate constants for THM hydrolysis were in the magnitude of 10(-2)‒10(-1) 1/h. When the boiled real tap water was stored in an enclosed container, THMs continued increasing during the first few hours and then kept decreasing later on due to the competition between hydrolysis and further formation. The removal of THMs, especially brominated THMs, by hydrolysis would greatly reduce one's exposure to disinfection by-products by consuming the boiled water stored in enclosed containers. PMID:25898249

  20. A proline-derived transannular N-cap for nucleation of short α-helical peptides.

    PubMed

    Tian, Yuan; Wang, Dongyuan; Li, Jingxu; Shi, Chuan; Zhao, Hui; Niu, Xiaogang; Li, Zigang

    2016-07-28

    We report herein a proline-derived transannular N-cap as a helix nucleating template in diverse bio-related peptide sequences via macrolactamization on resin. This approach takes advantage of synergistic stabilization effects of both N-capping properties of proline and substitution of a main chain hydrogen bond with a covalent bond. PMID:27357119

  1. Peptide library approach to uncover phosphomimetic inhibitors of the BRCA1 C-terminal domain.

    PubMed

    White, E Railey; Sun, Luxin; Ma, Zhong; Beckta, Jason M; Danzig, Brittany A; Hacker, David E; Huie, Melissa; Williams, David C; Edwards, Ross A; Valerie, Kristoffer; Glover, J N Mark; Hartman, Matthew C T

    2015-05-15

    Many intracellular protein-protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein-protein interactions mediated by serine phosphorylation. PMID:25654734

  2. The bioactive composite film prepared from bacterial cellulose and modified by hydrolyzed gelatin peptide.

    PubMed

    Lin, Shih-Bin; Chen, Chia-Che; Chen, Li-Chen; Chen, Hui-Huang

    2015-05-01

    The hydrolyzed gelatin peptides, obtained from the hydrolysis of Tilapia nilotica skin gelatin with alcalase and pronase E, were fractionated using an ultrafiltration system into hydrolyzed gelatin peptides-a (10 kDa membrane), hydrolyzed gelatin peptides-b1, and hydrolyzed gelatin peptides-b2 (5 kDa membrane) fractions. The highest oxygen radical absorbance capacity was observed in hydrolyzed gelatin peptides-b2, which contained more nonpolar amino acids than the other hydrolyzed gelatin peptides. Hydrolyzed gelatin peptides-b2 at a concentration of 12.5 mg/ml exhibited the highest proliferation ability and increased the expression of Type I procollagen mRNA, which indicated an enhanced collagen synthesis. Hydrolyzed gelatin peptides protected Detroit 551 cells from 2,2'-azobis(2-amidinopropane) dihydrochloride-induced oxidative damage and increased cell viability. Hydroxylpropylmethyl cellulose-modified bacterial cellulose and dried fabricated biofilm were less eligible for Detroit 551 cell proliferation than bacterial cellulose. The release of hydrolyzed gelatin peptides in bacterial cellulose film was slower than that in hydroxylpropylmethyl cellulose-modified bacterial cellulose and dried fabricated biofilm; thus, bacterial cellulose film and hydroxylpropylmethyl cellulose-modified bacterial cellulose and dried fabricated biofilm are suitable candidates for applications in delayed release type and rapid release type biofilms, respectively. PMID:25614493

  3. Peptide Library Approach to Uncover Phosphomimetic Inhibitors of the BRCA1 C-Terminal Domain

    PubMed Central

    White, E. Railey; Sun, Luxin; Ma, Zhong; Beckta, Jason M.; Danzig, Brittany A.; Hacker, David E.; Huie, Melissa; Williams, David C.; Edwards, Ross A.; Valerie, Kristoffer; Mark Glover, J. N.; Hartman, Matthew C. T.

    2015-01-01

    Many intracellular protein–protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein–protein interactions mediated by serine phosphorylation. PMID:25654734

  4. Synthesis of Hydrogen-Bond Surrogate α-helices as Inhibitors of Protein-Protein Interactions

    PubMed Central

    Miller, Stephen E.; Thomson, Paul F.; Arora, Paramjit S.

    2014-01-01

    The α-helix is a prevalent secondary structure in proteins and critical in mediating protein-protein interactions (PPIs). Peptide mimetics that adopt stable helices have become powerful tools for the modulation of PPIs in vitro and in vivo. Hydrogen-bond surrogate (HBS) α-helices utilize a covalent bond in place of an N-terminal i to i+4 hydrogen bond and have been used to target and disrupt PPIs that become dysregulated in disease states. These compounds have improved conformational stability and cellular uptake as compared to their linear peptide counterparts. The protocol presented here describes current methodology for the synthesis of HBS α-helical mimetics. The solid phase synthesis of HBS helices involves solid phase peptide synthesis with three key steps involving incorporation of N-allyl functionality within the backbone of the peptide, coupling of a secondary amine, and a ring-closing metathesis step. PMID:24903885

  5. Insulin C-peptide test

    MedlinePlus

    C-peptide ... the test depends on the reason for the C-peptide measurement. Ask your health care provider if ... C-peptide is measured to tell the difference between insulin produced by the body and insulin injected ...

  6. Exploitation of the Ornithine Effect Enhances Characterization of Stapled and Cyclic Peptides

    NASA Astrophysics Data System (ADS)

    Crittenden, Christopher M.; Parker, W. Ryan; Jenner, Zachary B.; Bruns, Kerry A.; Akin, Lucas D.; McGee, William M.; Ciccimaro, Eugene; Brodbelt, Jennifer S.

    2016-02-01

    A method to facilitate the characterization of stapled or cyclic peptides is reported via an arginine-selective derivatization strategy coupled with MS/MS analysis. Arginine residues are converted to ornithine residues through a deguanidination reaction that installs a highly selectively cleavable site in peptides. Upon activation by CID or UVPD, the ornithine residue cyclizes to promote cleavage of the adjacent amide bond. This Arg-specific process offers a unique strategy for site-selective ring opening of stapled and cyclic peptides. Upon activation of each derivatized peptide, site-specific backbone cleavage at the ornithine residue results in two complementary products: the lactam ring-containing portion of the peptide and the amine-containing portion. The deguanidination process not only provides a specific marker site that initiates fragmentation of the peptide but also offers a means to unlock the staple and differentiate isobaric stapled peptides.

  7. Exploitation of the Ornithine Effect Enhances Characterization of Stapled and Cyclic Peptides

    NASA Astrophysics Data System (ADS)

    Crittenden, Christopher M.; Parker, W. Ryan; Jenner, Zachary B.; Bruns, Kerry A.; Akin, Lucas D.; McGee, William M.; Ciccimaro, Eugene; Brodbelt, Jennifer S.

    2016-05-01

    A method to facilitate the characterization of stapled or cyclic peptides is reported via an arginine-selective derivatization strategy coupled with MS/MS analysis. Arginine residues are converted to ornithine residues through a deguanidination reaction that installs a highly selectively cleavable site in peptides. Upon activation by CID or UVPD, the ornithine residue cyclizes to promote cleavage of the adjacent amide bond. This Arg-specific process offers a unique strategy for site-selective ring opening of stapled and cyclic peptides. Upon activation of each derivatized peptide, site-specific backbone cleavage at the ornithine residue results in two complementary products: the lactam ring-containing portion of the peptide and the amine-containing portion. The deguanidination process not only provides a specific marker site that initiates fragmentation of the peptide but also offers a means to unlock the staple and differentiate isobaric stapled peptides.

  8. Mechanism of the myosin catalyzed hydrolysis of ATP as rationalized by molecular modeling

    PubMed Central

    Grigorenko, Bella L.; Rogov, Alexander V.; Topol, Igor A.; Burt, Stanley K.; Martinez, Hugo M.; Nemukhin, Alexander V.

    2007-01-01

    The intrinsic chemical reaction of adenosine triphosphate (ATP) hydrolysis catalyzed by myosin is modeled by using a combined quantum mechanics and molecular mechanics (QM/MM) methodology that achieves a near ab initio representation of the entire model. Starting with coordinates derived from the heavy atoms of the crystal structure (Protein Data Bank ID code 1VOM) in which myosin is bound to the ATP analog ADP·VO4−, a minimum-energy path is found for the transformation ATP + H2O → ADP + Pi that is characterized by two distinct events: (i) a low activation-energy cleavage of the PγOβγ bond and separation of the γ-phosphate from ADP and (ii) the formation of the inorganic phosphate as a consequence of proton transfers mediated by two water molecules and assisted by the Glu-459–Arg-238 salt bridge of the protein. The minimum-energy model of the enzyme–substrate complex features a stable hydrogen-bonding network in which the lytic water is positioned favorably for a nucleophilic attack of the ATP γ-phosphate and for the transfer of a proton to stably bound second water. In addition, the PγOβγ bond has become significantly longer than in the unbound state of the ATP and thus is predisposed to cleavage. The modeled transformation is viewed as the part of the overall hydrolysis reaction occurring in the closed enzyme pocket after ATP is bound tightly to myosin and before conformational changes preceding release of inorganic phosphate. PMID:17438284

  9. Influence of salt bridge interactions on the gas-phase stability of DNA/peptide complexes

    NASA Astrophysics Data System (ADS)

    Alves, Sandra; Woods, Amina; Delvolvé, Alice; Tabet, Jean Claude

    2008-12-01

    Negative ion mode electrospray ionization mass spectrometry was used to study DNA duplexes-peptide interaction. In the present study, we show that peptides that contain two adjacent basic residues interact noncovalently with DNA single strand or duplex. Fragmentation of the complexes between peptides containing basic residues and DNA were studied under collisions and showed unexpected dissociation pathways, as previously reported for peptide-peptide interactions. The binary complexes are dissociated either along fragmentation of the covalent bonds of the peptide backbone and/or along the single DNA strand backbone cleavage without disruption of noncovalent interaction, which demonstrates the strong binding of peptide to the DNA strand. Sequential MS/MS and MSn were further performed on ternary complexes formed between duplexes and peptides to investigate the nature of interaction. The CID spectra showed as major pathway the disruption of the noncovalent interactions and the formation of binary complexes and single-strand ions, directed by the nucleic acid gas-phase acidity. Indeed, a preferential formation of complexes with thymidine containing single strands is observed. An alternative pathway is also detected, in which complexes are dissociated along the covalent bond of the peptide and/or DNA according to the basicity. Our experimental data suggest the presence of strong salt bridge interactions between DNA and peptides containing basic residues.

  10. Succinimidyl Ester Surface Chemistry: Implications of the Competition between Aminolysis and Hydrolysis on Covalent Protein Immobilization

    PubMed Central

    2015-01-01

    N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins. PMID:25317495

  11. Assessment of taste attributes of peanut meal enzymatic-hydrolysis hydrolysates using an electronic tongue.

    PubMed

    Wang, Li; Niu, Qunfeng; Hui, Yanbo; Jin, Huali; Chen, Shengsheng

    2015-01-01

    Peanut meal is the byproduct of high-temperature peanut oil extraction; it is mainly composed of proteins, which have complex tastes after enzymatic hydrolysis to free amino acids and small peptides. The enzymatic hydrolysis method was adopted by using two compound proteases of trypsin and flavorzyme to hydrolyze peanut meal aiming to provide a flavor base. Hence, it is necessary to assess the taste attributes and assign definite taste scores of peanut meal double enzymatic hydrolysis hydrolysates (DEH). Conventionally, sensory analysis is used to assess taste intensity in DEH. However, it has disadvantages because it is expensive and laborious. Hence, in this study, both taste attributes and taste scores of peanut meal DEH were evaluated using an electronic tongue. In this regard, the response characteristics of the electronic tongue to the DEH samples and standard five taste samples were researched to qualitatively assess the taste attributes using PCA and DFA. PLS and RBF neural network (RBFNN) quantitative prediction models were employed to compare predictive abilities and to correlate results obtained from the electronic tongue and sensory analysis, respectively. The results showed that all prediction models had good correlations between the predicted scores from electronic tongue and those obtained from sensory analysis. The PLS and RBFNN prediction models constructed using the voltage response values from the sensors exhibited higher correlation and prediction ability than that of principal components. As compared with the taste performance by PLS model, that of RBFNN models was better. This study exhibits potential advantages and a concise objective taste assessment tool using the electronic tongue in the assessment of DEH taste attributes in the food industry. PMID:25985162

  12. Assessment of Taste Attributes of Peanut Meal Enzymatic-Hydrolysis Hydrolysates Using an Electronic Tongue

    PubMed Central

    Wang, Li; Niu, Qunfeng; Hui, Yanbo; Jin, Huali; Chen, Shengsheng

    2015-01-01

    Peanut meal is the byproduct of high-temperature peanut oil extraction; it is mainly composed of proteins, which have complex tastes after enzymatic hydrolysis to free amino acids and small peptides. The enzymatic hydrolysis method was adopted by using two compound proteases of trypsin and flavorzyme to hydrolyze peanut meal aiming to provide a flavor base. Hence, it is necessary to assess the taste attributes and assign definite taste scores of peanut meal double enzymatic hydrolysis hydrolysates (DEH). Conventionally, sensory analysis is used to assess taste intensity in DEH. However, it has disadvantages because it is expensive and laborious. Hence, in this study, both taste attributes and taste scores of peanut meal DEH were evaluated using an electronic tongue. In this regard, the response characteristics of the electronic tongue to the DEH samples and standard five taste samples were researched to qualitatively assess the taste attributes using PCA and DFA. PLS and RBF neural network (RBFNN) quantitative prediction models were employed to compare predictive abilities and to correlate results obtained from the electronic tongue and sensory analysis, respectively. The results showed that all prediction models had good correlations between the predicted scores from electronic tongue and those obtained from sensory analysis. The PLS and RBFNN prediction models constructed using the voltage response values from the sensors exhibited higher correlation and prediction ability than that of principal components. As compared with the taste performance by PLS model, that of RBFNN models was better. This study exhibits potential advantages and a concise objective taste assessment tool using the electronic tongue in the assessment of DEH taste attributes in the food industry. PMID:25985162

  13. Bacteriocin Inducer Peptides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel peptides produced by bacteriocin-producing bacteria stimulate the production of bacteriocins in vitro. The producer bacteria are cultured in the presence of a novel inducer bacteria and a peptide having a carboxy terminal sequence of VKGLT in order to achieve an increase in bacteriocin produc...

  14. Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process.

    PubMed

    Welderufael, Fisseha Tesfay; Gibson, Trevor; Methven, Lisa; Jauregi, Paula

    2012-10-15

    The overall aim of this work was to characterise the major angiotensin-converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilisation of β-lactoglobulin and casein-derived peptides (CDP) from whey. The adsorbed proteins were hydrolysed in situ, which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC(50)=287 μg/mL) and (ii) from β-lactoglobulin (IC(50)=128 μg/mL). The well-known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterised in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the bitterness of the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC(50)). PMID:23442643

  15. Weak bond screening system

    NASA Astrophysics Data System (ADS)

    Chuang, S. Y.; Chang, F. H.; Bell, J. R.

    Consideration is given to the development of a weak bond screening system which is based on the utilization of a high power ultrasonic (HPU) technique. The instrumentation of the prototype bond strength screening system is described, and the adhesively bonded specimens used in the system developmental effort are detailed. Test results obtained from these specimens are presented in terms of bond strength and level of high power ultrasound irradiation. The following observations were made: (1) for Al/Al specimens, 2.6 sec of HPU irradiation will screen weak bond conditions due to improper preparation of bonding surfaces; (2) for composite/composite specimens, 2.0 sec of HPU irradiation will disrupt weak bonds due to under-cured conditions; (3) for Al honeycomb core with composite skin structure, 3.5 sec of HPU irradiation will disrupt weak bonds due to bad adhesive or oils contamination of bonding surfaces; and (4) for Nomex honeycomb with Al skin structure, 1.3 sec of HPU irradiation will disrupt weak bonds due to bad adhesive.

  16. Optimization of enzymatic hydrolysis of shrimp waste for recovery of antioxidant activity rich protein isolate.

    PubMed

    Sowmya, R; Ravikumar, T M; Vivek, R; Rathinaraj, K; Sachindra, N M

    2014-11-01

    Shrimp waste is an important source of astaxanthin, which occur as a complex with proteins, and protein isolates as well as carotenoids are known to possess antioxidant activity. Investigations were carried out to optimize hydrolysis of shrimp waste using a bacterial protease to obtain antioxidant activity rich protein isolate. The effect of three process variables namely enzyme concentration to waste, incubation temperature and time on carotenoid recovery, protein content, trichloro acetic acid (TCA) soluble peptide content and DiPhenyl Picryl Hydrazylchloride (DPPH) scavenging activity was evaluated using a fractionally factorial design. A high correlation coefficient (>0.90) between the observed and the predicted values indicated the appropriateness of the design employed. Maximum carotenoid recovery was obtained by hydrolysing the shrimp waste with 0.3 % enzyme for 4 h. DPPH radical scavenging activity of carotenoprotein isolate was markedly affected by enzyme concentration, temperature and time of hydrolysis. The study indicated that in order to obtain the carotenoprotein from shrimp waste with higher carotenoid content hydrolysing with an enzyme concentration of 0.2-0.4 %, at lower temperature of 25-30° upto 4 h is ideal. However, in order to obtain the protein isolate with increased antioxidant activity hydrolysing at higher temperature of 50 °C, with higher enzyme concentration of 0.5 % for shorter duration is more ideal. PMID:26396312

  17. Hydrolysis of somatostatin by human tissue kallikrein after the amino acid pair phe-Phe.

    PubMed Central

    Pimenta, D C; Juliano, M A; Juliano, L

    1997-01-01

    Somatostatin-(1-14) was hydrolysed by human tissue kallikrein at the Phe7-Trp8 bond, after a Phe-Phe pair of amino acids, with similar kinetic parameters to those described for human high- and low-molecular-mass kininogens. Substance P and human insulin, which also contain a Phe-Phe pair in their sequences, were both resistant. More details of this hydrolytic specificity of human tissue kallikrein were obtained by synthesizing and assaying internally quenched fluorescent peptides containing the sequence of somatostatin-(1-14), as well as the reactive-centre loop of human kallikrein-binding protein (kallistatin). We also observed that human tissue kallikrein hydrolysed growth hormone-releasing hormone at the Arg11-Lys12 bond, although this peptide contains in its structure a pair of leucines (Leu22-Leu23), in contrast with the Phe-Phe pair in somatostatin. We have also demonstrated the susceptibility to human tissue kallikrein of some chromogenic peptide s with the general structure of X-Phe-Phe-p-nitroanilide and D-Pro-Phe-Phe-4-methylcoumaryl-7-amide. PMID:9355730

  18. Interactions of antimitotic peptides and depsipeptides with tubulin.

    PubMed

    Hamel, Ernest

    2002-01-01

    Tubulin is the target for an ever increasing number of structurally unusual peptides and depsipeptides isolated from a wide range of organisms. Since tubulin is the subunit protein of microtubules, the compounds are usually potently toxic to mammalian cells. Without exception, these (depsi)peptides disrupt cellular microtubules and prevent spindle formation. This causes cells to accumulate at the G2/M phase of the cell cycle through inhibition of mitosis. In biochemical assays, the compounds inhibit microtubule assembly from tubulin and suppress microtubule dynamics at low concentrations. Most of the (depsi)peptides inhibit the binding of Catharanthus alkaloids to tubulin in a noncompetitive manner, GTP hydrolysis by tubulin, and nucleotide turnover at the exchangeable GTP site on beta-tubulin. In general, the (depsi)peptides induce the formation of tubulin oligomers of aberrant morphology. In all cases tubulin rings appear to be formed, but these rings differ in diameter, depending on the (depsi)peptide present during their formation. PMID:12385035

  19. Alkaline Phosphatase-Mimicking Peptide Nanofibers for Osteogenic Differentiation.

    PubMed

    Gulseren, Gulcihan; Yasa, I Ceren; Ustahuseyin, Oya; Tekin, E Deniz; Tekinay, Ayse B; Guler, Mustafa O

    2015-07-13

    Recognition of molecules and regulation of extracellular matrix synthesis are some of the functions of enzymes in addition to their catalytic activity. While a diverse array of enzyme-like materials have been developed, these efforts have largely been confined to the imitation of the chemical structure and catalytic activity of the enzymes, and it is unclear whether enzyme-mimetic molecules can also be used to replicate the matrix-regulatory roles ordinarily performed by natural enzymes. Self-assembled peptide nanofibers can provide multifunctional enzyme-mimetic properties, as the active sequences of the target enzymes can be directly incorporated into the peptides. Here, we report enhanced bone regeneration efficiency through peptide nanofibers carrying both catalytic and matrix-regulatory functions of alkaline phosphatase, a versatile enzyme that plays a critical role in bone formation by regulating phosphate homeostasis and calcifiable bone matrix formation. Histidine presenting peptide nanostructures were developed to function as phosphatases. These molecules are able to catalyze phosphate hydrolysis and serve as bone-like nodule inducing scaffolds. Alkaline phosphatase-like peptide nanofibers enabled osteogenesis for both osteoblast-like and mesenchymal cell lines. PMID:26039144

  20. Alpha-helical stabilization by side chain shielding of backbone hydrogen bonds.

    PubMed

    García, Angel E; Sanbonmatsu, Kevin Y

    2002-03-01

    We study atomic models of the thermodynamics of the structural transition of peptides that form alpha-helices. The effect of sequence variation on alpha-helix formation for alanine-rich peptides, Ac-Ala21-methyl amide (A21) and Ac-A5 (AAARA)3A-methyl amide (Fs peptide), is investigated by atomic simulation studies of the thermodynamics of the helix-coil transition in explicit water. The simulations show that the guanidinium group in the Arg side chains in the Fs peptide interacts with the carbonyl group four amino acids upstream in the chain and desolvates backbone hydrogen bonds. This desolvation can be directly correlated with a higher probability of hydrogen bond formation. We find that Fs has higher helical content than A21 at all temperatures. A small modification in the amber force field reproduces the experimental helical content and helix-coil transition temperatures for the Fs peptide. PMID:11867710

  1. Antimicrobial Peptides from Fish

    PubMed Central

    Masso-Silva, Jorge A.; Diamond, Gill

    2014-01-01

    Antimicrobial peptides (AMPs) are found widely distributed through Nature, and participate in the innate host defense of each species. Fish are a great source of these peptides, as they express all of the major classes of AMPs, including defensins, cathelicidins, hepcidins, histone-derived peptides, and a fish-specific class of the cecropin family, called piscidins. As with other species, the fish peptides exhibit broad-spectrum antimicrobial activity, killing both fish and human pathogens. They are also immunomodulatory, and their genes are highly responsive to microbes and innate immuno-stimulatory molecules. Recent research has demonstrated that some of the unique properties of fish peptides, including their ability to act even in very high salt concentrations, make them good potential targets for development as therapeutic antimicrobials. Further, the stimulation of their gene expression by exogenous factors could be useful in preventing pathogenic microbes in aquaculture. PMID:24594555

  2. [The use of enzymatic hydrolysis for isolation of barbituric acid derivatives from blood (as exemplified by phenobarbital and barbamyl)].

    PubMed

    Chuvina, N A; Kolupaeva, A S; Strelova, O Iu; Zabolotskaia, I V; Gorbacheva, T V

    2010-01-01

    Modern isolation techniques by direct extraction with organic solvents or after protein precipitation by various sedimenting or salting-out agents are characterized by low efficiency and do not permit to liberate derivatives of barbituric acid from their complexes with blood proteins. The use of enzymatic hydrolysis makes it possible to break bonds between barbiturates and protein and thereby improve the efficiency of isolation. We performed enzymatic hydrolysis of the model phenobarbital-blood and barbamyl-blood complexes with the use of trypsin, pepsin, chymotrypsin, and papain. The degree of phenobarbital extraction with trypsin and barbamyl was estimated at 62.1 +/- 1.2% and 75.1 +/- 1.6% respectively; in other words, it was 32.7 +/- 1.0% and 51.1 +/- 1.0% higher than that achieved by traditional methods. Certain validation characteristics of the new method are presented. PMID:21265178

  3. Effects of ultrasonic treatment during acid hydrolysis on the yield, particle size and structure of cellulose nanocrystals.

    PubMed

    Guo, Juan; Guo, Xuxia; Wang, Siqun; Yin, Yafang

    2016-01-01

    Ultrasonic treatment is useful for the isolation of cellulose nanocrystals (CNCs). However, the effects of ultrasonic treatment on the structure and properties of CNCs have not been fully understood. We statistically analyzed the variations in yields and dimensions of CNCs prepared using acid hydrolysis, with or without ultrasonic treatment, and illustrated these variations by investigating the CNC structures. The results demonstrated that ultrasonic treatment promoted an increase in yields only for short hydrolysis times, while resulting in CNCs with shorter lengths and narrower dimension distributions. Furthermore, a prolonged acid attack under ultrasonic treatment caused a partial dissociation of cellulose hydrogen bond networks in the CNCs, ultimately resulting in the delamination and disorder of the cellulose crystalline structure, thus leading to a decrease in the width and thickness of the CNCs. This work provides more insights into the mechanisms of ultrasonic treatment on the structure and properties of CNCs. PMID:26453875

  4. Functionalization of poly(ε-caprolactone) surface with lactose-modified chitosan via alkaline hydrolysis: ToF-SIMS characterization.

    PubMed

    Tortora, Luca; Concolato, Sofia; Urbini, Marco; Giannitelli, Sara Maria; Basoli, Francesco; Rainer, Alberto; Trombetta, Marcella; Orsini, Monica; Mozetic, Pamela

    2016-06-01

    Functionalization of poly(ε-caprolactone) (PCL) was performed via hydrolysis and subsequent grafting of lactose-modified chitosan (chitlac) at two different degrees of derivatization (9% and 64%). Time of flight secondary ion mass spectrometry (ToF-SIMS) and multivariate analysis (principal component analysis) were successfully applied to the characterization of PCL surface chemistry, evidencing changes in the biopolymer surface following base-catalyzed hydrolysis treatment. ToF-SIMS analysis also confirmed positive EDC/NHS-catalyzed (EDC: N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide; NHS: N-hydroxysuccinimide) immobilization of chitlac onto activated PCL surface, with formation of amide bonds between PCL surface carboxyl groups and amine residues of chitlac. Yield of grafting reaction was also shown to be dependent upon the lactosilation degree of chitlac. PMID:26905217

  5. Ultrafast hydrolysis of a Lewis photoacid.

    PubMed

    Henrich, Joseph D; Suchyta, Scott; Kohler, Bern

    2015-02-12

    This study explores the concept that electronic excitation can dramatically enhance Lewis acidity. Specifically, it is shown that photoexcitation transforms an electron-deficient organic compound of negligible Lewis acidity in its electronic ground state into a potent excited-state Lewis acid that releases a proton from a nearby water molecule in 3.1 ps. It was shown previously (Peon et al. J. Phys. Chem. A 2001, 105, 5768) that the excited state of methyl viologen (MV(2+)) is quenched rapidly in aqueous solution with the formation of an unidentified photoproduct. In this study, the quenching mechanism and the identity of the photoproduct were investigated by the femtosecond transient absorption and fluorescence upconversion techniques. Transient absorption signals at UV probe wavelengths reveal a long-lived species with a pH-dependent lifetime due to reaction with hydronium ions at a bimolecular rate of 3.1 × 10(9) M(-1) s(-1). This species is revealed to be a charge-transfer complex consisting of a ground-state MV(2+) ion and a hydroxide ion formed when a water molecule transfers a proton to the bulk solvent. Formation of a contact ion pair between MV(2+) and hydroxide shifts the absorption spectrum of the former ion by a few nm to longer wavelengths, yielding a transient absorption spectrum with a distinctive triangle wave appearance. The slight shift of this spectrum, which is in excellent agreement with steady-state difference spectra recorded for MV(2+) at high pH, is consistent with an ion pair but not with a covalent adduct (pseudobase). The long lifetime of the ion pair at neutral pH indicates that dissociation occurs many orders of magnitude more slowly than predicted by the Smoluchowski-Debye equation. Remarkably, there is no evidence of geminate recombination, suggesting that the proton that is transferred to the solvent is conducted at least several water shells away. Although the hydrolysis mechanism has yet to be fully established, evidence suggests

  6. Bonded semiconductor substrate

    DOEpatents

    Atwater, Jr.; Harry A. , Zahler; James M.

    2010-07-13

    Ge/Si and other nonsilicon film heterostructures are formed by hydrogen-induced exfoliation of the Ge film which is wafer bonded to a cheaper substrate, such as Si. A thin, single-crystal layer of Ge is transferred to Si substrate. The bond at the interface of the Ge/Si heterostructures is covalent to ensure good thermal contact, mechanical strength, and to enable the formation of an ohmic contact between the Si substrate and Ge layers. To accomplish this type of bond, hydrophobic wafer bonding is used, because as the invention demonstrates the hydrogen-surface-terminating species that facilitate van der Waals bonding evolves at temperatures above 600.degree. C. into covalent bonding in hydrophobically bound Ge/Si layer transferred systems.

  7. Stimuli-responsive self-assembling peptides made from antibacterial peptides

    NASA Astrophysics Data System (ADS)

    Liu, Yanfei; Yang, Yanlian; Wang, Chen; Zhao, Xiaojun

    2013-06-01

    How to use bioactive peptide sequences as fundamental building blocks to make hydrogel materials which are stimuli-responsive? In this article, we provide a novel designed peptide comprising two antibacterial peptide sequences (KIGAKI)3-NH2 and a central tetrapeptide linker. Results show that balancing the forces of the electrostatic repulsion of the charged lysine residues against the hydrophobic collapse of the isoleucine and alanine residues and backbone β-sheet hydrogen bonding allows the structural transition and formation of individually dispersed nanofibers. Circular Dichroism (CD) and rheology analysis demonstrated that the designed peptide can undergo an abrupt structural transition from a random coil to a stable unimolecular β-hairpin conformation and subsequently form an elastic hydrogel when exposed to external stimuli such as pH, ionic strength and heat. The assembly kinetics of the obtained antibacterial sequence comprising peptide (ASCP) was studied by time-lapse Atomic Force Microscopy (AFM) and Thioflavin T (ThT) binding assay. In addition, the inherent antibacterial activity of the peptide hydrogel was confirmed by the antibacterial assay against Escherichia coli. This example described epitomizes the use of bioactive peptide sequences in the design of finite self-assembled structures with potential inherent activity. These hydrogel materials may find applications in drug delivery, tissue engineering and regenerative medicine.How to use bioactive peptide sequences as fundamental building blocks to make hydrogel materials which are stimuli-responsive? In this article, we provide a novel designed peptide comprising two antibacterial peptide sequences (KIGAKI)3-NH2 and a central tetrapeptide linker. Results show that balancing the forces of the electrostatic repulsion of the charged lysine residues against the hydrophobic collapse of the isoleucine and alanine residues and backbone β-sheet hydrogen bonding allows the structural transition and

  8. Molecular Dynamical Study on Ion Channeling through Peptide Nanotube

    NASA Astrophysics Data System (ADS)

    Sumiya, Norihito; Igami, Daiki; Takeda, Kyozaburo

    2011-12-01

    We theoretically study the possibility of ion channeling through peptide nanotubes (PNTs). After designing the minimal peptide nanorings (PNRs) and their aggregated form (peptide nanotubes, PNT) computationally, we carry out molecular dynamics (MD) calculations for cation channeling. The present MD calculations show that cation channeling through PNTs occurs. Furthermore, inter-ring hydrogen bonds (HBs) survive and maintain the tubular form of PNTs during cation channeling. We introduce mobility such that cation channeling can be evaluated quantitatively. As the ionic radius of the cation becomes smaller, the effective relaxation time τ becomes larger. Accordingly, mobilities of 10-2˜10-3[cm2/volt/sec] are calculated. In contrast, when an anion (F-) passes through the PNT, the inter-ring HBs are broken, thus inducing breakdown of the peptide backbone. Consequently, H atoms from the broken HBs surround the channeling anion (F-) and halt its motion.

  9. Sequence-Specific DNA Binding by a Short Peptide Dimer

    NASA Astrophysics Data System (ADS)

    Talanian, Robert V.; McKnight, C. James; Kim, Peter S.

    1990-08-01

    A recently described class of DNA binding proteins is characterized by the "bZIP" motif, which consists of a basic region that contacts DNA and an adjacent "leucine zipper" that mediates protein dimerization. A peptide model for the basic region of the yeast transcriptional activator GCN4 has been developed in which the leucine zipper has been replaced by a disulfide bond. The 34-residue peptide dimer, but not the reduced monomer, binds DNA with nanomolar affinity at 4^circC. DNA binding is sequence-specific as judged by deoxyribonuclease I footprinting. Circular dichroism spectroscopy suggests that the peptide adopts a helical structure when bound to DNA. These results demonstrate directly that the GCN4 basic region is sufficient for sequence-specific DNA binding and suggest that a major function of the GCN4 leucine zipper is simply to mediate protein dimerization. Our approach provides a strategy for the design of short sequence-specific DNA binding peptides.

  10. Steric-electronic effects in malarial peptides inducing sterile immunity

    SciTech Connect

    Moreno-Vranich, Armando; Patarroyo, Manuel E.

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer Is it evident that the residues position are relevant regarding of {phi} angular value. Black-Right-Pointing-Pointer The geometry considered for detailing the alterations undergone by HABPs. Black-Right-Pointing-Pointer The inter planar interactions ruled by clashes between the atoms making them up. -- Abstract: Conserved Plasmodium falciparum high activity binding peptides' (HABPs) most relevant proteins involved in malaria parasite invasion are immunologically silent; critical binding residues must therefore be specifically replaced to render them highly immunogenic and protection-inducing. Such changes have a tremendous impact on these peptides' steric-electronic effects, such as modifications to peptide length peptide bonds and electronic orbitals' disposition, to allow a better fit into immune system MHCII molecules and better interaction with the TCR which might account for the final immunological outcome.

  11. Evidence for Surface Recognition by a Cholesterol-Recognition Peptide.

    PubMed

    Mukai, Masaru; Glover, Kerney Jebrell; Regen, Steven L

    2016-06-21

    Two cholesterol recognition/interaction amino-acid consensus peptides, N-acetyl-LWYIKC-amide, and N-acetyl-CLWYIK-amide, have been coupled to exchangeable mimics of Chol (cholesterol) and Phos (1,2-dipalmitoyl-sn-glycerol-3-phospho-(1'rac-glycerol)) via disulfide bond formation. Equilibration between Chol and Phos via thiolate-disulfide interchange reactions has revealed that both peptides favor Chol as a nearest-neighbor in liquid-disordered (ld) bilayers to the same extent. In contrast, no Chol- or Phos-recognition could be detected by these peptides in analogous liquid-ordered (lo) bilayers. Fluorescence measurements of the tryptophan moiety have shown that both peptides favor the membrane-water interface. Taken together, these results provide strong evidence that the recognition behavior of the LWYIK motif is, fundamentally, a surface phenomenon but that partial penetration into the bilayer is also necessary. PMID:27283494

  12. Energy pulse bonding

    NASA Technical Reports Server (NTRS)

    Smith, G. C.

    1972-01-01

    To eliminate many of the present termination problems a technique called energy pulse bonding (EPB) was developed. The process demonstrated the capability of: (1) joining conductors without prior removal of insulations, (2) joining conductors without danger of brittle intermetallics, (3) increased joint temperature capability, (4) simultaneous formation of several bonds, (5) capability of higher joint density, and (6) a production oriented process. The following metals were successfully bonded in the solid state: copper, beryllium copper, phosphor bronze, aluminum, brass, and Kovar.

  13. BONDING ALUMINUM METALS

    DOEpatents

    Noland, R.A.; Walker, D.E.

    1961-06-13

    A process is given for bonding aluminum to aluminum. Silicon powder is applied to at least one of the two surfaces of the two elements to be bonded, the two elements are assembled and rubbed against each other at room temperature whereby any oxide film is ruptured by the silicon crystals in the interface; thereafter heat and pressure are applied whereby an aluminum-silicon alloy is formed, squeezed out from the interface together with any oxide film, and the elements are bonded.

  14. Chemical bonding technology

    NASA Technical Reports Server (NTRS)

    Plueddemann, E.

    1986-01-01

    Primers employed in bonding together the various material interfaces in a photovoltaic module are being developed. The approach develops interfacial adhesion by generating actual chemical bonds between the various materials bonded together. The current status of the program is described along with the progress toward developing two general purpose primers for ethylene vinyl acetate (EVA), one for glass and metals, and another for plastic films.

  15. Detection of trans–cis flips and peptide-plane flips in protein structures

    PubMed Central

    Touw, Wouter G.; Joosten, Robbie P.; Vriend, Gert

    2015-01-01

    A coordinate-based method is presented to detect peptide bonds that need correction either by a peptide-plane flip or by a trans–cis inversion of the peptide bond. When applied to the whole Protein Data Bank, the method predicts 4617 trans–cis flips and many thousands of hitherto unknown peptide-plane flips. A few examples are highlighted for which a correction of the peptide-plane geometry leads to a correction of the understanding of the structure–function relation. All data, including 1088 manually validated cases, are freely available and the method is available from a web server, a web-service interface and through WHAT_CHECK. PMID:26249342

  16. Valence bond entanglement entropy.

    PubMed

    Alet, Fabien; Capponi, Sylvain; Laflorencie, Nicolas; Mambrini, Matthieu

    2007-09-14

    We introduce for SU(2) quantum spin systems the valence bond entanglement entropy as a counting of valence bond spin singlets shared by two subsystems. For a large class of antiferromagnetic systems, it can be calculated in all dimensions with quantum Monte Carlo simulations in the valence bond basis. We show numerically that this quantity displays all features of the von Neumann entanglement entropy for several one-dimensional systems. For two-dimensional Heisenberg models, we find a strict area law for a valence bond solid state and multiplicative logarithmic corrections for the Néel phase. PMID:17930468

  17. Inhibition of aggregation of amyloid peptides by beta-sheet breaker peptides and their binding affinity.

    PubMed

    Viet, Man Hoang; Ngo, Son Tung; Lam, Nguyen Sy; Li, Mai Suan

    2011-06-01

    The effects of beta-sheet breaker peptides KLVFF and LPFFD on the oligomerization of amyloid peptides were studied by all-atom simulations. It was found that LPFFD interferes the aggregation of Aβ(16-22) peptides to a greater extent than does KLVFF. Using the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method, we found that the former binds more strongly to Aβ(16-22). Therefore, by simulations, we have clarified the relationship between aggregation rates and binding affinity: the stronger the ligand binding, the slower the oligomerization process. The binding affinity of pentapeptides to full-length peptide Aβ(1-40) and its mature fibrils has been considered using the Autodock and MM-PBSA methods. The hydrophobic interaction between ligands and receptors plays a more important role for association than does hydrogen bonding. The influence of beta-sheet breaker peptides on the secondary structures of monomer Aβ(1-40) was studied in detail, and it turns out that, in their presence, the total beta-sheet content can be enhanced. However, the aggregation can be slowed because the beta-content is reduced in fibril-prone regions. Both pentapeptides strongly bind to monomer Aβ(1-40), as well as to mature fibrils, but KLVFF displays a lower binding affinity than LPFFD. Our findings are in accord with earlier experiments that both of these peptides can serve as prominent inhibitors. In addition, we predict that LPFFD inhibits/degrades the fibrillogenesis of full-length amyloid peptides better than KLVFF. This is probably related to a difference in their total hydrophobicities in that the higher the hydrophobicity, the lower the inhibitory capacity. The GROMOS96 43a1 force field with explicit water and the force field proposed by Morris et al. (Morris et al. J. Comput. Chem. 1998, 19, 1639 ) were employed for all-atom molecular dynamics simulations and Autodock experiments, respectively. PMID:21563780

  18. Enzymatic hydrolysis of biomass from wood.

    PubMed

    Álvarez, Consolación; Reyes-Sosa, Francisco Manuel; Díez, Bruno

    2016-03-01

    Current research and development in cellulosic ethanol production has been focused mainly on agricultural residues and dedicated energy crops such as corn stover and switchgrass; however, woody biomass remains a very important feedstock for ethanol production. The precise composition of hemicellulose in the wood is strongly dependent on the plant species, therefore different types of enzymes are needed based on hemicellulose complexity and type of pretreatment. In general, hardwood species have much lower recalcitrance to enzymes than softwood. For hardwood, xylanases, beta-xylosidases and xyloglucanases are the main hemicellulases involved in degradation of the hemicellulose backbone, while for softwood the effect of mannanases and beta-mannosidases is more relevant. Furthermore, there are different key accessory enzymes involved in removing the hemicellulosic fraction and increasing accessibility of cellulases to the cellulose fibres improving the hydrolysis process. A diversity of enzymatic cocktails has been tested using from low to high densities of biomass (2-20% total solids) and a broad range of results has been obtained. The performance of recently developed commercial cocktails on hardwoods and softwoods will enable a further step for the commercialization of fuel ethanol from wood. PMID:26833542

  19. Synthesis, hydrolysis and stability of psilocin glucuronide.

    PubMed

    Martin, Rafaela; Schürenkamp, Jennifer; Pfeiffer, Heidi; Lehr, Matthias; Köhler, Helga

    2014-04-01

    A two-step synthesis of psilocin glucuronide (PCG), the main metabolite of psilocin, with methyl 2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-α-d-glucopyranuronate is reported. With the synthesized PCG, hydrolysis conditions in serum and urine were optimized. Escherichia coli proved to be a better enzyme source for β-glucuronidase than Helix pomatia. It was essential to add ascorbic acid to serum samples to protect psilocin during incubation. Furthermore the stability of PCG and psilocin was compared as stability data are the basis for forensic interpretation of measurements. PCG showed a greater long-term stability after six months in deep frozen serum and urine samples than psilocin. The short-term stability of PCG for one week in whole blood at room temperature and in deep frozen samples was also better than that of psilocin. Therefore, PCG can be considered to be more stable than the labile psilocin and should always be included if psilocin is analyzed in samples. PMID:24513688

  20. Catalytic Activities Of [GADV]-Peptides

    NASA Astrophysics Data System (ADS)

    Oba, Takae; Fukushima, Jun; Maruyama, Masako; Iwamoto, Ryoko; Ikehara, Kenji

    2005-10-01

    We have previously postulated a novel hypothesis for the origin of life, assuming that life on the earth originated from “[GADV]-protein world”, not from the “RNA world” (see Ikehara's review, 2002). The [GADV]-protein world is constituted from peptides and proteins with random sequences of four amino acids (glycine [G], alanine [A], aspartic acid [D] and valine [V]), which accumulated by pseudo-replication of the [GADV]-proteins. To obtain evidence for the hypothesis, we produced [GADV]-peptides by repeated heat-drying of the amino acids for 30 cycles ([GADV]-P30) and examined whether the peptides have some catalytic activities or not. From the results, it was found that the [GADV]-P30 can hydrolyze several kinds of chemical bonds in molecules, such as umbelliferyl-β-D-galactoside, glycine-p-nitroanilide and bovine serum albumin. This suggests that [GADV]-P30 could play an important role in the accumulation of [GADV]-proteins through pseudo-replication, leading to the emergence of life. We further show that [GADV]-octapaptides with random sequences, but containing no cyclic compounds as diketepiperazines, have catalytic activity, hydrolyzing peptide bonds in a natural protein, bovine serum albumin. The catalytic activity of the octapeptides was much higher than the [GADV]-P30 produced through repeated heat-drying treatments. These results also support the [GADV]-protein-world hypothesis of the origin of life (see Ikehara's review, 2002). Possible steps for the emergence of life on the primitive earth are presented.