Isoelectric Point (isoelectric + point)

Distribution by Scientific Domains
Distribution within Chemistry

Kinds of Isoelectric Point

  • different isoelectric point

  • Selected Abstracts

    Isoelectric points of viruses

    B. Michen
    Summary Viruses as well as other (bio-)colloids possess a pH-dependent surface charge in polar media such as water. This electrostatic charge determines the mobility of the soft particle in an electric field and thus governs its colloidal behaviour which plays a major role in virus sorption processes. The pH value at which the net surface charge switches its sign is referred to as the isoelectric point (abbreviations: pI or IEP) and is a characteristic parameter of the virion in equilibrium with its environmental water chemistry. Here, we review the IEP measurements of viruses that replicate in hosts of kingdom plantae, bacteria and animalia. IEPs of viruses are found in pH range from 19 to 84; most frequently, they are measured in a band of 35 < IEP < 7. However, the data appear to be scattered widely within single virus species. This discrepancy is discussed and should be considered when IEP values are used to account for virus sorption processes. [source]

    Gadolinium, a mechano-sensitive channel blocker, inhibits osmosis-initiated motility of sea- and freshwater fish sperm, but does not affect human or ascidian sperm motility

    CYTOSKELETON, Issue 4 2003
    Zoltn Krasznai
    Abstract Exposure to hypo-osmotic or hyperosmotic environment triggers the initiation of fish sperm motility. In this article, we report that calcium and potassium channel blockers do not influence motility of puffer fish sperm but calmodulin antagonists reversibly decrease it, suggesting that calmodulin,Ca2+ interactions are prerequisite for the initiation of sperm motility in this species. Gadolinium (a stretch activated ion channel blocker) decreased the motility of puffer fish sperm from 92 3% to 6 3% and that of carp sperm from 91 7% to 3.5 4.3% in a dose-dependent manner (10,40 ,M). The effect of gadolinium was reversible, suggesting that stretch activated ion channels participate in the initiation of sperm motility of the two species. Gadolinium inhibits changes in the isoelectric point of certain proteins of puffer fish sperm, which occur when sperm motility is initiated in a hypertonic solution. Anisotropy measurements showed that hypo-osmotic treatment, which initiates carp sperm motility, increased membrane fluidity. When hypo-osmotic treatment was given in the presence of gadolinium, the sperm membrane remained as rigid as in quiescent cells, while motility was blocked. By contrast, gadolinium did not influence the motility parameters of Ciona or human sperm. Based on these lines of evidence, we suggest that conformational changes of mechanosensitive membrane proteins are involved in osmolality-dependent but not osmolality-independent sperm. Cell Motil. Cytoskeleton 55:232,243, 2003. 2003 Wiley-Liss, Inc. [source]

    Insulin analogues: an example of applied medical science

    B. Sheldon
    Insulin analogues were developed to try and achieve more physiological insulin replacement from injection in the subcutaneous site. Their pharmacokinetics and pharmacodynamics differ from human insulin when injected subcutaneously because of alterations in the amino acid sequence of the insulin molecule. The rapid-acting insulin analogues, lispro, aspart and glulisine, have a rapid onset of action and shorter duration of action because of changes to the B26,30 portion of insulin inhibiting formation of dimers and hexamers. They appear to improve postprandial glucose, incidence of hypoglycaemia and patient satisfaction and, when used in combination with basal insulin analogues, improve glycosylated haemoglobin in comparison to conventional insulin therapy. Additionally, they have been successfully used in children, pregnant women, in pump therapy and as part of premixed biphasic regimens. The two basal insulin analogues, glargine and detemir, developed by adjusting the isoelectric point and adding a fatty acid residue, respectively, have a protracted duration of action and a relatively smooth profile. Their pharmacokinetic and pharmacodynamic profiles have been assessed using euglycaemic clamp protocols. Both analogues have a longer duration of action, less of a peak of activity and a reduced variability with repeated injection. There is some evidence to suggest that detemir may have a slight hepatoselective effect. Clinical studies have shown a lower relative risk of hypoglycaemia and detemir appears to have a weight-sparing action. Insulin analogues represent a successful example of applied medical science. [source]

    Age-dependent variations of cell response to oxidative stress: Proteomic approach to protein expression and phosphorylation

    ELECTROPHORESIS, Issue 14 2005
    Yuri Miura Dr.
    Abstract We investigated the protein profiles of variously aged rat astrocytes in response to oxidative stress. After H2O2 -exposure of cells at 100,M for 30,min, the relative intensity of ten protein spots changed on two-dimensional (2-D) gels compared with control gels after silver staining. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis after in-gel digestion revealed that six of these spots corresponded to three kinds of proteins, each of which was composed of a protein and its modified form with a different isoelectric point (pI). These three proteins were identified as peroxiredoxins (PRDXs) II and III, and calpactin I light chain (p11). H2O2 -exposure increased the intensity of the spot with lower pI and simultaneously decreased that of the spot with higher pI for both PRDXs II and III. In addition, the expression of annexin VII, S -adenosyl- L -homocysteine hydrolase, elongation factor II fragment (EF-II), and adenosine deaminase was increased by H2O2 -exposure in astrocytes from variously aged rats. Using the Pro-Q Diamond staining, heat shock protein 60,kDa (Hsp 60) and ,-tubulin were observed to be phosphorylated upon H2O2 -exposure. While phosphorylation of ,-tubulin was correlated positively with age, the changes in abundance of ten protein spots as described above were independent of age. These results suggest that aging does not suppress the responses aimed at limiting injury and promoting repair brought about by severe oxidative stress, and might affect cell dynamics including the formation of microtubules. [source]

    Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations

    ELECTROPHORESIS, Issue 7-8 2004
    Yi Zhu
    Abstract Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E. coli). Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential. A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis. The result is a pI versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains. [source]

    Purification and cDNA Cloning of Lysozyme II from Cabbage Butterfly, Artogeia rapae Larvae

    BANG In Seok
    ABSTRACT Last instar larvae of cabbage butterfly Artogeia rapae respond to injection of bacteria with a set of inducible antibacterial peptides/proteins. The inducible peptides/proteins are related to the known hinnavins (I and II) and lysozymes (I and II). The lysozyme II has been isolated by heat treatment, cation exchange, and reversed-phase chromatography from immunized hemolymph of last instar larvae. The lysozyme II gene of A. rapae was isolated and its nucleotide sequence was determined by the RACE-PCR from immunized fat body with E. coli. It has an open reading frame of 414 bp nucleotide corresponding to 138 amino acids including an 18 amino acid signal sequence. The molecular weight and the isoelectric point of Artogeia lysozyme II without a signal peptide were 13,649.38 Da and 9.11, respectively. It is great similarity with Manduca lysozyme among other lepidopteran. [source]

    Proteomic Identification of the Involvement of the Mitochondrial Rieske Protein in Epilepsy

    EPILEPSIA, Issue 3 2005
    Heike Junker
    Summary:,Purpose: Kindled seizures are widely used to model epileptogenesis, but the molecular mechanisms underlying the attainment of kindling status are largely unknown. Recently we showed that achievement of kindling status in the Sprague,Dawley rat is associated with a critical developmental interval of 25 1 days; the identification of this long, well-defined developmental interval for inducing kindling status makes possible a dissection of the cellular and genetic events underlying this phenomenon and its relation to normal and pathologic brain function. Methods: By using proteomics on cerebral tissue from our new rat kindling model, we undertook a global analysis of protein expression in kindled animals. Some of the identified proteins were further investigated by using immunohistochemistry. Results: We report the identification of a modified variant of the Rieske iron-sulfur protein, a component of the mitochondrial cytochrome bc1 complex, whose isoelectric point is shifted toward more alkaline values in the hippocampus of kindled rats. By immunohistochemistry, the Rieske protein is well expressed in the hippocampus, except in the CA1 subfield, an area of selective vulnerability to seizures in humans and animal models. We also noted an asymmetric, selective expression of the Rieske protein in the subgranular neurons of the dorsal dentate gyrus, a region implicated in neurogenesis. Conclusions: These results indicate that the Rieske protein may play a role in the response of neurons to seizure activity and could give important new insights into the molecular pathogenesis of epilepsy. [source]

    Bovine Serum Albumin and Lysozyme Adsorption on Calcium Phosphate Particles

    Berit Mueller
    Two model proteins that are oppositely charged at neutral pH , bovine serum albumin (BSA) and lysozyme, with acidic and alkaline isoelectric points, respectively , are used to investigate the protein adsorption behaviour of hydroxyapatite and beta-tricalcium phosphate (, -TCP) particles. Both calcium phosphate based particles are highly relevant for the fabrication of bioactive and resorbable bone implants. The investigations are carried out by combining zeta potential and Vis spectroscopy measurements. The changes of zeta potential and isoelectric point are determined as a function of added protein. Both proteins form a monolayer on , -TCP, while on hydroxyapatite only semi-monolayers were measured. For BSA, a side-on adsorption mode is suggested, whereas end-on adsorption appears to be most likely for lysozyme. The zeta potential curves as a function of adsorbed protein show that plateaus of the protein amounts adsorbed increase with charge saturation. In addition, the spatial charge distribution of both proteins is modelled to get a further understanding of the initial adsorption orientation of the biomolecules, supporting the findings from the experimental data. The reported findings can be transferred to the adsorption behaviour of a variety of proteins on calcium phosphate surfaces and are helpful for the fabrication of bone-analogous calcium phosphate/protein nanocomposites. [source]

    Completing the hypusine pathway in Plasmodium

    FEBS JOURNAL, Issue 20 2009
    Deoxyhypusine hydroxylase is an E-Z type HEAT repeat protein
    In searching for new targets for antimalarials we investigated the biosynthesis of hypusine present in eukaryotic initiation factor-5A (eIF-5A) in Plasmodium. Here, we describe the cloning and expression of deoxyhypusine hydroxylase (DOHH), which completes the modification of eIF-5A through hydroxylation of deoxyhypusine. The dohh cDNA sequence revealed an ORF of 1236 bp encoding a protein of 412 amino acids with a calculated molecular mass of 46.45 kDa and an isoelectric point of 4.96. Interestingly, DOHH from Plasmodium has a FASTA SCORE of only 27 compared with its human ortholog and contains several matches similar to E-Z-type HEAT-like repeat proteins (IPR004155 (InterPro), PF03130 (Pfam), SM00567 (SMART) present in the phycocyanin lyase subunits of cyanobacteria. Purified DOHH protein displayed hydroxylase activity in a novel in vitro DOHH assay, but phycocyanin lyase activity was absent. dohh is present as a single-copy gene and is transcribed in the asexual blood stages of the parasite. A signal peptide at the N-terminus might direct the protein to a different cellular compartment. During evolution, Plasmodium falciparum acquired an apicoplast that lost its photosynthetic function. It is possible that plasmodial DOHH arose from an E/F-type phycobilin lyase that gained a new role in hydroxylation. Structured digital abstract ,,MINT-7255047: DHS (uniprotkb:P49366) enzymaticly reacts (MI:0414) with eIF-5A (uniprotkb:Q710D1) by enzymatic studies (MI:0415) ,,MINT-7255326: DOHH (uniprotkb:Q8I701) enzymaticly reacts (MI:0414) with eIF-5A (uniprotkb:Q710D1) by enzymatic studies (MI:0415) [source]

    Mass spectrometric detection of tyrosine sulfation in human pancreatic trypsinogens, but not in tumor-associated trypsinogen

    FEBS JOURNAL, Issue 2 2008
    Outi Itkonen
    Trypsinogen-1 and -2 are well-characterized enzymes that are expressed in the pancreas and also in several other tissues. Many cancers produce trypsinogen isoenzymes that differ from the pancreatic ones with respect to substrate specificity and isoelectric point. These tumor-associated trypsinogens play a pivotal role in cancer progression and metastasis. The differences between these and the pancreatic isoenzymes have been suggested to be caused by post-translational modification, either sulfation or phosphorylation of a tyrosine residue. We aimed to elucidate the cause of these differences. We isolated trypsinogens from pancreatic juice and conditioned medium from a colon carcinoma cell line. Intact proteins, and tryptic and chymotryptic peptides were characterized by electrospray ionization mass spectrometry. We also used immunoblotting with antibody against phosphotyrosine and N-terminal sequencing. The results show that pancreatic trypsinogen-1 and -2 are sulfated at Tyr154, whereas tumor-associated trypsinogen-2 is not. Detachment of a labile sulfogroup could be demonstrated by both in-source dissociation and low-energy collision-induced dissociation in a tandem mass spectrometer. Tyrosine sulfation is an ubiquitous protein modification occurring in the secretory pathway, but its significance is often underestimated due to difficulties in its analysis. Sulfation is an almost irreversible modification that is thought to regulate protein,protein interactions and the activity of proteolytic enzymes. We conclude that the previously known differences in charge, substrate specificity and inhibitor binding between pancreatic and tumor-associated trypsinogens are probably caused by sulfation of Tyr154 in pancreatic trypsinogens. [source]

    Two novel Mesocestoides vogae fatty acid binding proteins , functional and evolutionary implications

    FEBS JOURNAL, Issue 1 2008
    Gabriela Alvite
    This work describes two new fatty acid binding proteins (FABPs) identified in the parasite platyhelminth Mesocestoides vogae (syn. corti). The corresponding polypeptide chains share 62% identical residues and overall 90% similarity according to clustalx default conditions. Compared with Cestoda FABPs, these proteins share the highest similarity score with the Taenia solium protein. M. vogae FABPs are also phylogenetically related to the FABP3/FABP4 mammalian FABP subfamilies. The native proteins were purified by chromatographical procedures, and apparent molecular mass and isoelectric point were determined. Immunolocalization studies determined the localization of the expression of these proteins in the larval form of the parasite. The genomic exon,intron organization of both genes is also reported, and supports new insights on intron evolution. Consensus motifs involved in splicing were identified. [source]

    Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp.

    FEBS JOURNAL, Issue 7 2004
    SIB1 in cold-adaptation
    A psychrotrophic bacterium Shewanella sp. strain SIB1 was grown at 4 and 20 C, and total soluble proteins extracted from the cells were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of these patterns showed that the cellular content of a protein with a molecular mass of 28 kDa and an isoelectric point of four greatly increased at 4 C compared to that at 20 C. Determination of the N-terminal amino acid sequence, followed by the cloning and sequencing of the gene encoding this protein, revealed that this protein is a member of the FKBP family of proteins with an amino acid sequence identity of 56% to Escherichia coli FKBP22. This protein was overproduced in E. coli in a His-tagged form, purified, and analyzed for peptidyl-prolyl cis-trans isomerase activity. When this activity was determined by the protease coupling assay using N -succinyl-Ala-Leu-Pro-Phe- p -nitroanilide as a substrate at various temperatures, the protein exhibited the highest activity at 10 C with a kcat/Km value of 0.87 m,1s,1. When the peptidyl-prolyl cis-trans isomerase activity was determined by the RNase T1 refolding assay at 10 and 20 C, the protein exhibited higher activity at 10 C with a kcat/Km value of 0.50 m,1s,1. These kcat/Km values are lower but comparable to those of E. coli FKBP22. We propose that a FKBP family protein is involved in cold-adaptation of psychrotrophic bacteria. [source]

    Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvacea

    FEBS JOURNAL, Issue 2 2004
    Shicheng Chen
    We have isolated a laccase (lac1) from culture fluid of Volvariella volvacea, grown in a defined medium containing 150 m CuSO4, by ion-exchange and gel filtration chromatography. Lac1 has a molecular mass of 58 kDa as determined by SDS/PAGE and an isoelectric point of 3.7. Degenerate primers based on the N-terminal sequence of purified lac1 and a conserved copper-binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full-length cDNA clones. The cDNA of lac1 contained an ORF of 1557 bp encoding 519 amino acids. The amino acid sequence from Ala25 to Asp41 corresponded to the N-terminal sequence of the purified protein. The first 24 amino acids are presumed to be a signal peptide. The expression of lac1 is regulated at the transcription level by copper and various aromatic compounds. RT-PCR analysis of gene transcription in fungal mycelia grown on rice-straw revealed that, apart from during the early stages of substrate colonization, lac1 was expressed at every stage of the mushroom developmental cycle defined in this study, although the levels of transcription varied considerably depending upon the developmental phase. Transcription of lac1 increased sharply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body morphogenesis. Gene expression then declined to ,,20,30% of peak levels throughout the subsequent stages of sporophore development. [source]

    Numerical calculations of the pH of maximal protein stability

    FEBS JOURNAL, Issue 1 2004
    The effect of the sequence composition, three-dimensional structure
    A large number of proteins, found experimentally to have different optimum pH of maximal stability, were studied to reveal the basic principles of their preferenence for a particular pH. The pH-dependent free energy of folding was modeled numerically as a function of pH as well as the net charge of the protein. The optimum pH was determined in the numerical calculations as the pH of the minimum free energy of folding. The experimental data for the pH of maximal stability (experimental optimum pH) was reproducible (rmsd = 0.73). It was shown that the optimum pH results from two factors , amino acid composition and the organization of the titratable groups with the 3D structure. It was demonstrated that the optimum pH and isoelectric point could be quite different. In many cases, the optimum pH was found at a pH corresponding to a large net charge of the protein. At the same time, there was a tendency for proteins having acidic optimum pHs to have a base/acid ratio smaller than one and vice versa. The correlation between the optimum pH and base/acid ratio is significant if only buried groups are taken into account. It was shown that a protein that provides a favorable electrostatic environment for acids and disfavors the bases tends to have high optimum pH and vice versa. [source]

    Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-,-1,4-glucanase from blue mussel, Mytilus edulis

    FEBS JOURNAL, Issue 16 2000
    Bingze Xu
    A cellulase (endo-,-1,4- d -glucanase, EC from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 C. Another unusual feature is that the enzyme retains 55,60% of its maximum activity at 0 C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Vgtales, CNRS, Joseph Fourier Universit, Grenoble, France, personal communication). [source]

    Time Controlled Protein Release from Layer-by-Layer Assembled Multilayer Functionalized Agarose Hydrogels

    Sumit Mehrotra
    Abstract Axons of the adult central nervous system exhibit an extremely limited ability to regenerate after spinal cord injury. Experimentally generated patterns of axon growth are typically disorganized and randomly oriented. Support of linear axonal growth into spinal cord lesion sites has been demonstrated using arrays of uniaxial channels, templated with agarose hydrogel, and containing genetically engineered cells that secrete brain-derived neurotrophic factor (BDNF). However, immobilizing neurotrophic factors secreting cells within a scaffold is relatively cumbersome, and alternative strategies are needed to provide sustained release of BDNF from templated agarose scaffolds. Existing methods of loading the drug or protein into hydrogels cannot provide sustained release from templated agarose hydrogels. Alternatively, here it is shown that pH-responsive H-bonded poly(ethylene glycol)(PEG)/poly(acrylic acid)(PAA)/protein hybrid layer-by-layer (LbL) thin films, when prepared over agarose, provided sustained release of protein under physiological conditions for more than four weeks. Lysozyme, a protein similar in size and isoelectric point to BDNF, is released from the multilayers on the agarose and is biologically active during the earlier time points, with decreasing activity at later time points. This is the first demonstration of month-long sustained protein release from an agarose hydrogel, whereby the drug/protein is loaded separately from the agarose hydrogel fabrication process. [source]


    INSECT SCIENCE, Issue 1 2003
    LI Zhao-fei
    Abstract Ubiquitin (UBI) plays a very important role in regulated non-lysosomal ATP dependent protein degradation. In the present work, the coding sequence of Spodoptera litura UBI gene was isolated (GenBank Accession No. AF436066). The length of this ORF is 228bp, encoding a protein with Mr of 8.56 kD and isoelectric point of 6.56. Multiple sequence alignment indicated that S. litura UBI is very similar to the homologous proteins of other eukaryotic species and it has 84% identity with S. litura nucleopolyhedrovirus (SpltMNPV) UBI at amino acid level. RT-PCR analysis showed that S. litura UBI gene is ubiquitously expressed in larva tissues which are susceptible to SpltMNPV infection. By constructing E. coli expression vector, S. litura UBI was highly expressed and the recombinant protein was purified using Ni-NTA resin column, and currently further study on the function of S. litura UBI in SpltMNPV infection is underway. [source]

    Identification of four low molecular and water-soluble proteins from grape (Vitis vinifera L.) seeds

    Ting Zhou
    Summary Profiles of soluble proteins isolated from mature seeds of grape (Vitis vinifera L.) pomace were studied using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI,TOF,MS). Two-dimensional gels stained with Coomassie brilliant blue revealed more than fifty protein spots. Four abundant protein spots showing low molecular weight (Mr) and wide isoelectric point (pI) were analysed by MALDI,TOF,MS, resulting in their identification. Taken together, these results suggest that identified proteins may be linked to seed development and metabolism, but more instructive is that they have some potential functions for future food application. These results provide some insights into conversion of grape processing wastes into useful products or even as raw material for other industries. [source]

    Isoelectric points of viruses

    B. Michen
    Summary Viruses as well as other (bio-)colloids possess a pH-dependent surface charge in polar media such as water. This electrostatic charge determines the mobility of the soft particle in an electric field and thus governs its colloidal behaviour which plays a major role in virus sorption processes. The pH value at which the net surface charge switches its sign is referred to as the isoelectric point (abbreviations: pI or IEP) and is a characteristic parameter of the virion in equilibrium with its environmental water chemistry. Here, we review the IEP measurements of viruses that replicate in hosts of kingdom plantae, bacteria and animalia. IEPs of viruses are found in pH range from 19 to 84; most frequently, they are measured in a band of 35 < IEP < 7. However, the data appear to be scattered widely within single virus species. This discrepancy is discussed and should be considered when IEP values are used to account for virus sorption processes. [source]

    Bacillus pumilus SG2 isolated from saline conditions produces and secretes two chitinases

    G. Ahmadian
    Abstract Aims: Isolation and characterization of chitinases from a halotolerant Bacillus pumilus. Methods and Results: Bacillus pumilus strain SG2 was isolated from saline conditions. It is able to produce chitinase activity at high salt concentration. SDS-PAGE analysis of the B. pumilus SG2 culture supernatant showed two major bands that were induced by chitin. The amino acid sequence of the two proteins, designated ChiS and ChiL, showed a high homology with the chitinase of B. subtilis CHU26, and chitinase A of B. licheniformis, respectively. N -terminal signal peptide of both proteins was also determined. The molecular weight and isoelectric point of the chitinases were determined to be 63 and 74 kDa, and 45 and 51, for ChiS and ChiL respectively. The genes encoding for both chitinases were isolated and their sequence determined. The regulation of the chitinase genes is under the control of the catabolite repression system. Conclusions: Secreted chitinase genes and their flanking region on the genome of B. pumilus SG2 have been identified and sequenced. Significance and Impact of the Study: This is the first report of a multiple chitinases-producing B. pumilus halotolerant strain. We have identified two chitinases by using a reverse genetics approach. The chitinases show resistance to salt. [source]

    Penicillium chrysogenum glucose oxidase , a study on its antifungal effects

    . Leiter
    Abstract Aims:, Purification and characterization of the high molecular mass Candida albicans -killing protein secreted by Penicillium chrysogenum. Methods and Results:, The protein was purified by a combination of ultrafiltration, chromatofocusing and gel filtration. Enzymological characteristics [relative molecular mass (Mr) = 155 000, subunit structure ,2 with Mr,, = 76 000, isoelectric point (pI) = 54] were determined using SDS-PAGE and 2D-electrophoresis. N-terminal amino acid sequencing and homology search demonstrated that the antifungal protein was the glucose oxidase (GOX) of the fungus. The enzyme was cytotoxic for a series of bacteria, yeasts and filamentous fungi. Vitamin C (10 mg ml,1) prevented oxidative cell injuries triggered by 0004 U GOX in Emericella nidulans cultures but bovine liver catalase was ineffective even at a GOX : catalase activity ratio of 0004 : 200 U. A secondary inhibition of growth in E. nidulans cultures by the oxygen-depleting GOX,catalase system was likely to replace the primary inhibition exerted by H2O2. Conclusions:,Penicillium chrysogenum GOX possesses similar enzymological features to those described earlier for other Penicillium GOXs. Its cytotoxicity was dependent on the inherent antioxidant potential of the test micro-organisms. Significance and Impact of the Study:,Penicillium chrysogenum GOX may find future applications in glucose biosensor production, the disinfection of medical implants or in the food industry as an antimicrobial and/or preservative agent. [source]

    Encapsulation efficiency and release behaviors of bovine serum albumin loaded in alginate microspheres prepared by spraying

    Jie Zhang
    Abstract Spraying and spraying with an electrostatic field (SEF) were employed to prepare alginate microspheres for delivering proteins, especially for intestinal digestive enzymes and cytokines. The encapsulation efficiency (EE) of a model protein [bovine serum albumin (BSA)] at a pH value lower than the isoelectric point was 20% higher than that at a natural pH. Moreover, for the microspheres prepared by SEF, EE improved significantly with increasing electric voltage. The interactions between BSA and the alginate microspheres were identified with Fourier transform infrared spectroscopy. The release profiles in vitro showed a controlled and pH-responsive release manner for the encapsulated BSA. A first-order release equation was postulated and modified to describe the release kinetics with an obviously initial burst release related to the eroded porous matrix. The equation fit the release data well when the pH value and composition of the release media were changed. The analysis of the release kinetics indicated that the drug release rate was in an inverse ratio to the diameter of the microspheres. Increasing the gas flow rate or electric voltage decreased both the mean diameter and size distribution of the microspheres significantly and enhanced the release rate of loaded drugs from alginate microspheres. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis revealed that BSA kept its structural integrity during the encapsulation and release process. 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source]

    Preparation and adsorption behavior of a cellulose-based, mixed-mode adsorbent with a benzylamine ligand for expanded bed applications

    Dong Gao
    Abstract A novel mixed-mode expanded bed adsorbent with anion-exchange properties was explored with benzylamine as the functional ligand. The cellulose composite matrix, densified with stainless steel powder, was prepared with the method of water-in-oil suspension thermal regeneration. High activation levels of the cellulose matrix were obtained with allyl bromide because of the relative inertness of the allyl group under the conditions of the activation reaction. After the formation of the bromohydrin with N -bromosuccinimide and coupling with benzylamine, the activated matrix was derived to function as a mixed-mode adsorbent containing both hydrophobic and ionic groups. The protein adsorption capacity was investigated with bovine serum albumin as a model protein. The results indicated that the prepared adsorbent could bind bovine serum albumin with a high adsorption capacity, and it showed salt tolerance. Effective desorption was achieved by a pH adjustment across the isoelectric point of the protein. The interactions between the cell and adsorbent were studied, and the bioadhesion was shielded by the adjustment of the salt concentration above 0.1M. Stable fluidization in the expanded bed was obtained even in a 2% (dry weight) yeast suspension. The direct capture of target proteins from a biomass-containing feedstock without extra dilution steps could be expected with the mixed-mode adsorbent prepared in this work, and this would be especially appropriate for expanded bed adsorption applications. 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source]

    Extraction of native collagen from limed bovine split wastes through improved pretreatment methods

    Dong Li
    Abstract BACKGROUND: The large amount of limed bovine split wastes discharged by the leather industry has raised concerns regarding their environmental effect. The objective of this work was to perform pilot plant trials to extract high-value native collagen from these wastes through improved pretreatment methods. RESULTS: EDTA- and HCl-pretreatment gave similar removal percentages of inorganic substances. Owing to the open structure of fibers, the collagen yield of HCl-pretreated splits (HPS) (41.31%) was higher than that of EDTA-pretreated splits (EPS) (10.42%). Furthermore, HCl-pretreated split collagen (HPC) had a more acidic isoelectric point, lower content of primary amino groups, larger Z-average particle size and higher relative viscosity than EDTA-pretreated split collagen (EPC). Electrophoretic analysis and circular dichroism spectra revealed the maintenance of polypeptide and triple helix conformation, respectively. In addition, the transition temperatures of EPC (34.7 C) and HPC (34.6 C) detected by differential scanning calorimetry (DSC) were close to that of commercial collagen from calfskin (CCC) (35.7 C). CONCLUSION: A process of native collagen extraction from limed bovine split wastes was proposed. While both EPC and HPC represented similar physicochemical properties to those of CCC, the collagen yield of HPS was much higher than that of EPS. Copyright 2008 Society of Chemical Industry [source]

    Purification and Characterization of an ,-L-Rhamnosidase from Aspergillus terreus of Interest in Winemaking

    M.V. Gallego
    ABSTRACT: An enzyme with ,-L-rhamnosidase activity was purified to homogeneity from a culture filtrate of Aspergillus terreus after growth in a medium containing L-rhamnose as the sole carbon source. The biosynthesis of this enzyme was repressed by glucose. The enzyme had a molecular mass of 96 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 4.6 as determined by analytical isoelectric focusing. The pH and temperature optima for the enzyme were found to be 4.0 and 44 C, respectively. Using p-nitrophenyl-,-L-rhamnopyranoside as a substrate, the enzyme exhibited Michaelis-Menten kinetics with KM and Vmax values of 0.17 mM and 84 U/mg, respectively. The enzyme was inhibited competitively by L-rhamnose (K1 2.5 mM). Divalent cations such as Ca2+ Mg2+ Zn2+ and Co2+ stimulated the a-L-rhamnosidase activity, whereas this was inhibited by Hg2+ and Cd2+. Ethanol (12% v/v) and glucose (21% w/v) decreased enzyme activity by approximately 20%, while this was not affected by SO2. [source]

    An investigation of the factors controlling the adsorption of protein antigens to anionic PLG microparticles

    James Chesko
    Abstract This work examines physico-chemical properties influencing protein adsorption to anionic PLG microparticles and demonstrates the ability to bind and release vaccine antigens over a range of loads, pH values, and ionic strengths. Poly(lactide-co-glycolide) microparticles were synthesized by a w/o/w emulsification method in the presence of the anionic surfactant DSS (dioctyl sodium sulfosuccinate). Ovalbumin (OVA), carbonic anhydrase (CAN), lysozyme (LYZ), lactic acid dehydrogenase, bovine serum albumin (BSA), an HIV envelope glyocoprotein, and a Neisseria meningitidis B protein were adsorbed to the PLG microparticles, with binding efficiency, initial release and zeta potentials measured. Protein (antigen) binding to PLG microparticles was influenced by both electrostatic interaction and other mechanisms such as van der Waals forces. The protein binding capacity was directly proportional to the available surface area and may have a practical upper limit imposed by the formation of a complete protein monolayer as suggested by AFM images. The protein affinity for the PLG surface depended strongly on the isoelectric point (pI) and electrostatic forces, but also showed contributions from nonCoulombic interactions. Protein antigens were adsorbed on anionic PLG microparticles with varying degrees of efficiency under different conditions such as pH and ionic strength. Observable changes in zeta potentials and morphology suggest the formation of a surface monolayer. Antigen binding and release occur through a combination of electrostatic and van der Waals interactions occurring at the polymer-solution interface. 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:2510-2519, 2005 [source]

    Aminated gelatin as a nasal absorption enhancer for peptide drugs: evaluation of absorption enhancing effect and nasal mucosa perturbation in rats

    Jian Wang
    This study was carried out to evaluate the potential of aminated gelatin as a nasal absorption enhancer for peptide drugs. The absorption-enhancing effect was investigated in rats using insulin and fluorescein isothiocyanate-dextran with a molecular weight of 4.4 kDa (FD-4) as model drugs. The absorption of insulin was estimated by measuring the changes in plasma glucose levels following intranasal administration, and that of FD-4 was determined by measuring its plasma concentration after dosing. The hypoglycaemic effect after intranasal administration of insulin with aminated gelatin significantly increased compared with that after intranasal administration of insulin in phosphate buffered saline, indicating that aminated gelatin effectively enhanced the nasal absorption of insulin. In contrast, neither kind of native gelatin (isoelectric point = 5.0 and 9.0) showed any absorption-enhancing effect. The pH of the formulations and the concentration of aminated gelatin were found to affect the hypoglycaemic effect. In addition, aminated gelatin at a concentration of 0.2 % significantly enhanced the absorption and the efflux of FD-4 through the rat nasal mucosa. The possible perturbation of aminated gelatin to nasal mucosa was evaluated by measuring the leaching of lactate dehydrogenase (LDH) using an in-situ perfusion rat model. Aminated gelatin presented a concentration-dependent (0.1-0.4 %) but relatively small effect on the LDH leaching from the rat nasal epithelial membrane. These results suggest that positively charged aminated gelatin could be a new absorption enhancer for nasal delivery of peptide drugs. [source]

    Gelatin Microspheres as a Pulmonary Delivery System: Evaluation of Salmon Calcitonin Absorption

    The use of negatively and positively charged gelatin microspheres for pulmonary delivery of salmon calcitonin was examined in rats. The microspheres were prepared using acidic gelatin (isoelectric point (IEP):, 5.0) and basic gelatin (IEP, 9.0) for the negatively and positively charged microspheres, respectively. The average diameters of positively charged gelatin microspheres in the dry state were 3.4, 11.2, 22.5 and 71.5 ,m, and that of negatively charged gelatin microspheres was 10.9 ,m. Neither positively nor negatively charged gelatin microspheres underwent any degradation in pH 7.0 PBS and there was less than 8% degradation in bronchoalveolar lavage fluid (BALF) after 8 h. In in-vitro release studies in pH 7.0 PBS, salmon calcitonin was rapidly released from positively charged gelatin microspheres within 2 h, and its cumulative release was approximately 85%. In addition, the release profiles were not influenced by particle sizes. The release rates of salmon calcitonin from negatively charged gelatin microspheres were lower than that from positively charged gelatin microspheres. The cumulative release was approximately 40% after 2 h, but there was no evidence of any sustained release. The pulmonary absorption of salmon calcitonin from gelatin microspheres was estimated by measuring its hypocalcaemic effect in rats. The pharmacological availability after administration of salmon calcitonin in positively and negatively charged gelatin microspheres was significantly higher than that in pH 7.0 PBS. The pharmacological availability after administration of salmon calcitonin in positively charged gelatin microspheres was significantly higher than that in negatively charged gelatin microspheres. Administration of salmon calcitonin in positively charged gelatin microspheres with smaller particle sizes led to a higher pharmacological availability. The pharmacological availability after pulmonary administration of salmon calcitonin in positively charged gelatin microspheres with particle sizes of 3.4 and 11.2 ,m was approximately 50%. In conclusion, the gelatin microspheres have been shown to be a useful vehicle for pulmonary delivery of salmon calcitonin. [source]

    Aqueous Processing and Stabilization of Manganese Zinc Ferrite Powders via a Passivation,Dispersion Approach

    Michael M. Mandanas
    A dispersion scheme for aqueous processing of manganese zinc ferrite suspensions is presented. The addition of oxalic acid leads to the formation of a uniform negative charge on the surface such that a cationic polyelectrolyte, polyethyleneimine (PEI), adsorbs and provides electrosteric dispersion. At 0.5 w/w (weight percent with respect to the dry powder) oxalic acid addition, there is a relatively uniform negative surface charge (approximately ,30 mV) within the suspension pH range investigated (3,10), eliminating the isoelectric point (pH ,7.6) present for the as-received metal oxide powder. At the addition of 0.5 w/w PEI on an oxalate-treated surface, the surface charge is constant and positive (,20 mV) through a wide pH range, ,5,10. The resulting rheological data for passivation,dispersion of relatively high-solids manganese zinc ferrite suspensions (,80 wt%) demonstrate improved colloid stability with improved rheological properties. The resulting apparent viscosity and Bingham yield point is 0.01 Pas (12.0 cP) and 0.24 Pa (2.4 dynes/cm2), respectively. A sulfonated napthalene-based dispersant, typically used in industry, gives an apparent viscosity and Bingham yield point of 0.03 Pas (32 cP) and 3.1 Pa (31 dynes/cm2), respectively. [source]

    Aqueous Processing of Titanium Carbide Green Sheets

    Jing-Xian Zhang
    TiC sheets were prepared by an aqueous tape-casting process. The zeta potential measurement showed that the isoelectric point for TiC powders in the absence of dispersant had a pH value of ,3.3. According to the surface properties of TiC powders, a cationic polymer PEI was selected as dispersant. In the presence of dispersant, the isoelectric point increased to a pH value of ,10.4. The slip stability was determined by visual observation of the fluidity of the slip as well as the settling of the powders. Results showed that the amount of dispersant required to achieve a minimum of viscosity for 50 vol% suspensions was equal to 1.2 wt%. In the absence and presence of dispersant, stable slips could be obtained in the pH ranges 7,9 and 11,12, respectively. The rheological measurements showed that with PEI as dispersant, TiC suspensions exhibited a small time dependent behavior. With polyvinyl alcohol as binder and glycerol as plasticizer, suspensions showed a thixotropic feature. As-cast tapes were dried in air at room temperature. The results showed that it was possible to fabricate homogeneous green tapes with smooth surfaces from these suspensions. [source]