Low Molecular Weight Peak Research Articles

Fractionation and characterisation of pectin-rich extracts from garlic biomass

Polysaccharides from garlic waste leaf and skin biomass have been isolated using a sequential extraction protocol and characterised using constituent sugar composition and linkage analysis, spectroscopy, chromatography and dilute solution viscometry. The results revealed that the isolated polysaccharides were predominantly pectins. The predominant monosaccharide in all samples was galacturonic acid (>61 %), followed by galactose and rhamnose. The pectins extracted from skin biomass were mainly homogalacturonan (83–91 %), whereas those extracted from leaf biomass comprised both homogalacturonan (62–65 %) and rhamnogalacturonan-I (35–38 %). The degree of methyl esterification of uronic acids in all samples was 44–56 %. The peak molecular weight of the main polysaccharide population in each sample was ∼ 350 x103 g/mol, with leaf extracts and the skin acidic extract containing a second, lower molecular weight peak. Overall, waste garlic biomass is a potential resource for commercial pectin extraction for use in food or pharmaceutical industries.

Monoclonal Antibody Sequence Variants Disguised as Fragments: Identification, Characterization, and Their Removal by Purification Process Optimization

During early stage development of a therapeutic IgG1 monoclonal antibody, high levels of low molecular weight (LMW) peaks were observed by high performance size-exclusion chromatography and capillary electrophoresis. Further characterization of the LMW peak enriched HPSEC fractions using reversed phase liquid chromatography coupled to mass spectrometry showed these LMW species were 47 kDa and 50 kDa in size. However, the measured masses could not be matched to any fragments resulting from peptide bond hydrolysis. To identify these unknown LMW species, molecular characterization methods were employed, including high-throughput sequencing of RNA. Transcriptomic analysis revealed the LMW species were generated by mis-splicing events in the heavy chain transcript, which produced truncated heavy chain products that assembled with the light chain to mimic the appearance of fragments identified by routine purity assays. In an effort to improve product quality, an optimized purification process was developed. Characterization of the process intermediates confirmed removal of both LMW species by the optimized process. Our study demonstrates that deep-dive analytical characterization of biotherapeutics is critical to ensure product quality and inform process development. Transcriptomic analysis tools can help identify the cause of unknown species, and plays a key role in product and process characterization.

Effect of steam explosion on dietary fiber, polysaccharide, protein and physicochemical properties of okara

Okara is the byproduct of tofu and soymilk industries. A large amount of okara is produced annually in the world, but only a small amount is fully utilized. The effect of steam explosion (SE) treatment on dietary fiber, protein and physiochemical properties of okara was investigated in this paper. The results showed that untreated okara contained 76.38% total dietary fiber (TDF) and 18.10% protein, but the content of soluble dietary fiber (SDF) was only 1.34%. After SE treatment, SDF content increased significantly. When the SE strength was 1.5 MPa for 30s, SDF content increased to 36.28%, higher by 26 times compared to the control okara. However, the contents of TDF and SDF both decreased when the SE strength enhanced further. Gel filtration chromatography showed that molecular weight of okara polysaccharides were distributed from 55 KDa to 2087 KDa. With the increase of SE strength, the ratio of low molecular weight polysaccharides increased and the molecular weight range of polysaccharides became narrower. Okara protein showed two molecular weight peaks. After SE treatment, the area of high molecular weight peak decreased and low molecular weight peak increased. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that okara protein appeared in five bands: 73.7 KDa, 55 KDa, 41.1 KDa, 32.7 KDa and 20.2 KDa. After SE treatment, the bands of 73.7 KDa, 55 KDa and 41.1 KDa weakened markedly, indicating high SE strength can dissociate macromolecular protein aggregates. Water solubility of okara increased significantly, while swelling capacity, oil and water holding capacities decreased after SE treatment. Scanning electron microscopy (SEM) demonstrated that the loose sheet structure of okara surface was destroyed into small fragments after SE treatment and the internal nucleated structure was disintegrated into small particles at high SE strength. The change in morphological structure also produced important influences on physiochemical properties of okara. This study suggested that SE treatment is an effective method for improving SDF content and quality of okara, so as to expand its development and utilization.

Characterization of bovine serum albumin hydrolysates prepared by subcritical water processing.

The impact of subcritical water processing (SWP), in a temperature range of 240-300 °C, on the formation of hydrolysates from bovine serum albumin (BSA) was investigated in this study. SDS-PAGE analysis of the samples treated at all temperatures did not reveal any bands. Yield, as evaluated by the Kjeldahl and Biuret methods, varied and decreased beyond 280 °C (P < 0.05). The molecular mass of the hydrolysates was highly affected by temperature, with the formation of a new low molecular weight peak, as revealed by gel permeation chromatography. SWP at 280 °C caused the greatest release of free amino groups, as shown by the TNBS assay (P < 0.05). Regardless of the type of amino acids, the maximum amount was obtained at 280 °C. At this temperature, the generation of alanine and glycine was relatively high. The optimization of processing parameters may enhance the production of valuable peptides without the need for additional catalysts.

Purification and characterization of CRISP-3 from human seminal plasma and its real-time binding kinetics with PSP94.

Cysteine-rich secretory proteins (CRISPs) have been postulated to have a role in male reproduction and prostate pathophysiology. Of the mammalian CRISPs, CRISP-3 levels in particular have been shown to be upregulated in prostate cancer. Efforts have been made to obtain highly pure CRISP-3 for gaining structure-function information of this protein. However, well characterized and highly pure protein is not available yet. CRISPs from snake venom have been purified using prostate secretory protein of 94 amino acids (PSP94) has been reported earlier. In the present study, CRISP-3 was purified to homogeneity from human seminal plasma using human PSP94-immnobilized affinity column. The molecular mass of the purified protein was determined by SDS-PAGE followed by immunoblotting and found to be ∼26kDa and ∼28kDa. The purity was further verified using MALDI-TOF MS analysis, where two peaks at m/z 25509 and 27715 were obtained. The lower molecular weight peak corresponds to the calculated molecular mass of CRISP-3 (∼26kDa); whereas the higher molecular weight peak was confirmed to be the glycosylated form (∼28kDa) from the deglycosylation experiment. Binding of PSP94 in increasing concentrations to purified CRISP-3 immobilized chip was further validated using surface plasmon resonance. The kinetics data suggested that purified CRISP-3 binds specifically and with high affinity to PSP94. In conclusion, a homogeneous preparation of highly pure CRISP-3 protein is obtained from human seminal plasma.

NMP of styrene in batch and CSTR at elevated temperatures: Modeling experimental trends

Nitroxide-mediated polymerization (NMP) is conducted in batch and in a continuous stirred-tank reactor (CSTR) at temperatures up to 200°C using a thermally stable nitroxide. A kinetic model was developed using the Predici® software package to further understand the experimental trends under batch and continuous operation. Using the estimated activation/deactivation rate coefficients from the batch polymerization, the model predicts that a significant amount of alkoxyamine is exiting the CSTR under steady state conditions, consistent with a low molecular weight peak found experimentally. The predicted outlet concentration of alkoxyamine at steady state is decreased by increasing the temperature and residence time in the reactor. Batch chain extension of the polymer produced in the CSTR supports the potential for continuous production of block copolymers.

In-vitro study of the effects of oxidative degradants on thermoplastic polyurethanes

In-vitro study of the effects of oxidative degradants on thermoplastic polyurethanes

Regulation of Sarcoplasmic Reticulum Ca2+ ATPase 2 (SERCA2) Activity by Phosphodiesterase 3A (PDE3A) in Human Myocardium: PHOSPHORYLATION-DEPENDENT INTERACTION OF PDE3A1 WITH SERCA2

Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity.

Carbodiimide induced cross-linking, ligand addition, and degradation in gelatin.

The water-soluble carbodiimide, 1-ethyl-3-(3-(dimethylaminopropyl)-carbodiimide (EDC) is widely used in protein chemistry. We used EDC-induced gelatin cross-linking as a model for amide bond formation to resolve reaction ambiguities with common variables of buffers, gelatin concentration, and pH. Percentage changes in SEC high molecular weight peak areas were used to follow the reactions. Differences in reaction rate and extent were observed with four commonly used buffers, while differences in extent were observed for commonly used concentrations and pH. We also investigated an anhydride mechanism for aqueous EDC-induced amide bond formation that has received little attention since its proposal in 1995. Gelatin carboxyl groups had a synergistic role during the addition of hydrazine to corroborate the anhydride formation between carboxyl groups. EDC-induced degradation of gelatin was investigated using percentage changes in SEC low molecular weight peak areas. The degradation occurred in excess EDC at neutral to alkaline pH and was enhanced substantially when reacting amino groups were not available. A mechanism of EDC-induced gelatin degradation is proposed and designated the extended Khorana mechanism. This EDC side reaction has the potential to occur in peptides and proteins under similar conditions.

Distribution and Speciation of Iron and Zinc in Grain of Two Wheat Genotypes

This study aimed to determine differences among wheat cultivars in the distribution and speciation of Fe and Zn in grain milling fractions. Cultivars with higher Fe and Zn concentrations in the wholemeal flour were found to contain higher concentrations in the white flour. Soluble Fe and Zn were extracted and analyzed by size exclusion-inductively coupled plasma mass spectrometry. Fe speciation varied between milling fractions with a low molecular weight (LMW) complex likely to be Fe-deoxymugenic acid/nicotianamine being the predominant extractable Fe species in white flour, accounting for approximately 85% of the extractable Fe. Bran fractions had a lower amount of LMW-Fe form but more as soluble Fe-phytate and an unidentified high molecular weight peak. In the white flour fraction soluble Zn was found to be present mainly as a LMW peak likely to be Zn-nicotianamine complex. Soluble Fe-phytate was found in the white flour fraction of a high-Fe cultivar but not in a low-Fe cultivar.

Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol

Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803-G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP(-/-) mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG.

Properties of a novel type of starch found in the double mutant “sweet wheat”

The double mutant “sweet wheat” (SW), which produces substantial amounts of sugars in immature seeds, is missing two starch synthases, namely granule-bound starch synthase I (GBSSI) and starch synthase IIa (SSIIa). The lack of these two enzymes causes major changes in the attributes of SW seed, starch, and starch granules. SW seeds appear normal during early stages of development, but become shrunken when seeds begin to mature and dry. However, even in immature seed, starch granules are small and misshapen, and high levels of maltose are present throughout seed development. The crystallinity of SW starch is altered in that a major peak typical of the cereal A-type diffraction pattern is absent, and the gelatinization temperature of SW starch is considerably lower than that of wild-type starch. Amylopectin from SW seed has a substantially lower molecular weight than that from wild-type seed, and a low molecular weight peak with a bimodal distribution is found only in SW starch. This peak contains linear malto-oligosaccharides as well as short, branched glucans. SW starch has an increased proportion of branches with DP<10, and chains with DP 2 and 3 are particularly increased. These changes suggest that sweet wheat starch is being modified in an atypical manner by isoamylases and/or β-amylases.

Effects of dual initiators and catalytic additives on atom transfer radical polymerization of styrene

The effect of two initiators, so-called dual initiators system on atom radical transfer polymerization (ATRP), were studied with dimethyl-2,6-dibromohepanedioate (DMDBHD) and ethyl-2-bromoisobutyrate (EBIB). Cu(I)Br as catalyst and N,N,N′,N′,N′-pentamethyl-diethylenetriamine as ligand were employed for styrene ATRP. Interestingly, bimodal MWD were shown for the dual initiator system, and one of the peaks had higher molecular weight (MW) and the other had lower MW compared to a one-initiator system. The lower MW peak in bimodal peaks seemed to be mainly resulting from EBIB and the higher MW peak from DMDBHD. Furthermore, methylaluminoxane (MAO) was fed into the ATRP reaction to observe the effect of it on ATRP. As the MAO/CuBr molar ratio in feed increased from 0 to 1, the molecular weight and conversion increased without a notable change in PDI and curve shape of GPC. The conversion in the presence of MAO was also increased with the increase in MAO/CuBr molar ratio. The effect of Cp*TiCl3 on the ATRP was opposite to that of MAO. As the Cp*TiCl3/CuBr molar ratio increased from 0 to 1, the conversion of polymerization was down from 56 to 35%. Furthermore, the molecular weight was drastically decreased from 10,000 to 5,500, but their PDI did not show a significant change. These results can elucidated by the retarding effect of Cp*TiCl3 on the propagation of polymerization.

Characterization of Reaction Intermediate Aggregates in Aniline Oxidative Polymerization at Low Proton Concentration

Aggregates of reaction intermediates form during the early stages of aniline oxidative polymerization whenever the initial mole ratio of proton concentration to aniline monomer concentration is low ([H(+)](0)/[An](0) <or= 1.0). Detailed characterization is carried out on those aggregates. The intermediate aggregates show a UV-Vis absorption peak at around 410 nm when dispersed in aqueous solution, whereas the peak is centered on 370 nm when dissolved in an organic solvent such as N-methylpyrrolidone. The electronic band gap decreases when the intermediates aggregate to form a solid, and thus, the absorption peak is red-shifted. Gel permeation chromatography (GPC) shows the aggregates contain a major low molecular weight peak with a long tail. The oligoanilines with low molecular weights consistently show a UV-Vis absorption peak at around 370 nm. Mass spectrometry confirms that the intermediate aggregates contain mainly a component with mass number 363 (M + H(+)), likely a tetramer. UV-Vis, GPC, mass spectrometry, NMR, FTIR, and XRD characterization results are presented and chemical structures for the tetramer are proposed. The major components of the intermediate aggregates are likely highly symmetric phenazine- and dihydrophenazine-containing structures. These particular organic compounds have not been identified before as intermediates. The aggregation and precipitation of the tetramers apparently stabilizes these intermediates. The aggregates are highly crystalline, as evidenced by powder X-ray diffraction. A new reaction mechanism for the formation of these intermediates is proposed.

The Crystal Structure of Progesterone 5β-Reductase from Digitalis lanata Defines a Novel Class of Short Chain Dehydrogenases/Reductases

Progesterone 5beta-reductase (5beta-POR) catalyzes the stereospecific reduction of progesterone to 5beta-pregnane-3,20-dione and is a key enzyme in the biosynthetic pathway of cardenolides in Digitalis (foxglove) plants. Sequence considerations suggested that 5beta-POR is a member of the short chain dehydrogenase/reductase (SDR) family of proteins but at the same time revealed that the sequence motifs that in standard SDRs contain the catalytically important residues are missing. Here we present crystal structures of 5beta-POR from Digitalis lanata in complex with NADP(+) at 2.3A and without cofactor bound at 2.4A resolution together with a model of a ternary complex consisting of 5beta-POR, NADP(+), and progesterone. Indeed, 5beta-POR displays the fold of an extended SDR. The architecture of the active site is, however, unprecedented because none of the standard catalytic residues are structurally conserved. A tyrosine (Tyr-179) and a lysine residue (Lys-147) are present in the active site, but they are displayed from novel positions and are part of novel sequence motifs. Mutating Tyr-179 to either alanine or phenylalanine completely abolishes the enzymatic activity. We propose that the distinct topology reflects the fact that 5beta-POR reduces a conjugated double bond in a steroid substrate via a 1-4 addition mechanism and that this requires a repositioning of the catalytically important residues. Our observation that the sequence motifs that line the active site are conserved in a number of bacterial and plant enzymes of yet unknown function leads us to the proposition that 5beta-POR defines a novel class of SDRs.

Molecular mass characteristics of polyethylene produced with supported vanadium‐magnesium catalysts

AbstractHighly active supported vanadium‐magnesium catalysts (VMC) produce polyethylene (PE) with broad and bimodal molecular mass distribution (MMD) in comparison with the famous titanium‐magnesium catalysts (TMC). The effect of hydrogen as an efficient chain‐transfer agent on the MMD of PE has been studied. Increasing hydrogen concentration causes a considerable broadening of MMD of PE due to the shift of the low molecular weight peak on the MMD curve. At the same time, the high molecular weight shoulder stays at the same position even at high hydrogen concentration. This means that VMC contain two types of active centre. One type is very reactive in the chain‐transfer reaction with hydrogen. These centres produce low molecular weight PE in polymerization in the presence of hydrogen. The other type of active centre is not active in chain transfer with hydrogen. These centres produce high molecular weight PE ((1–3) × 106) and hydrogen does not affect the position of the high molecular weight shoulder. MMD data were used to analyze the kinetics of the chain‐transfer reaction with hydrogen and to calculate the rate constants of this reaction. Copyright © 2005 Society of Chemical Industry

Effect of Number of Carbons in Added Disulfide on Breadmaking Quality

ABSTRACTBreadmaking properties such as bread height and specific volume were improved in bread from wheat flour with added disulfides such as dimethy disulfide (M2S2), diethyl disulfide (E2S2), dipropyl disulfide (Pro2S2), and dibutyl disulfide (B2S2). However, the amount of disulfides for maximum breadmaking properties was various and strongly related to the number of carbons (C) in the disulfide. Brabender farinographs of disulfide‐wheat flour showed an interesting profile, and the properties of the modification of the width of the tail. Size‐exclusion high performance liquid chromatography (SE‐HPLC) of the wheat proteins in the control and the disulfide‐added flours showed profiles of low, medium, and high molecular weight peaks. The area of the high molecular weight peak was larger in the disulfide‐added flours than in the control, indicating that this protein was important for the improvement of breadmaking properties.

Modification of a Fischer‐Tropsch wax by grafting with maleic anhydride

AbstractThe polarity of a hard Fischer‐Tropsch paraffin wax was improved through grafting of maleic anhydride (MA) initiated by dibenzoyl peroxide (DBP), which is reflected by a significant increase in the polar component of the surface free energy. MA and DBP concentrations of 3 wt % each are sufficient for significant modification of the polarity of the wax. FTIR spectra confirm the presence of anhydride groups on the wax chains, while gel permeation chromatograms show the development of a low molecular weight peak, which may be indicative of wax degradation. The grafting of wax by MA decreases the specific enthalpy of melting, as determined through differential scanning calorimetry. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 662–668, 2004

Effect of Leek (A. ampeloprasum L.) and Scallion (A. chinense L.) on Breadmaking Properties

Allium such as leek (A. ampeloprasum L.) and scallion (A. chinense L.) powders blended with wheat flour improved the breadmaking properties of bread height (mm) and specific volume (cm3/g). Brabender farino-graphs of the Allium (leek and scallion) powder/wheat flour showed remarkable changes in the profile such as width of the tail. Size-exclusion high performance liquid chromatography (SE-HPLC) of the wheat proteins in the Allium (leek and scallion) blended or not blended wheat flour showed a profile of low, medium and high molecular weight peaks. The area of the high molecular weight peak of the wheat proteins was larger in the Allium blended flours than in control (wheat flour) indicating that these protein fractions were related to the improvement in breadmaking properties.

Effect of Welsh Onion (Allium fistulosum L.) on Breadmaking Properties

ABSTRACTBreadmaking properties, such as bread height and specific volume, were improved in bread, made with wheat flour blended with Welsh onion (Allium fistulosum L.) powder, which is high in di‐n‐propyl disulfide (Pro2S2). Breadmaking properties also were improved in bread prepared from wheat flour blended with Pro2S2 alone. Brabender farinographs of Welsh onion powder/wheat flour and Pro2S2/wheat flour showed interesting properties in modifying the width of the tail. Size‐exclusion high performance liquid chromatography of the wheat proteins in the control and blended flours showed a profile of low, medium, and high molecular weight peaks. The area of the high molecular weight peak was larger in the 2 blended flours than in control, indicating this protein was important for the improvement.