Diethylaminoethyl Ion-exchange Chromatography Research Articles

Lily bulb polyphenol oxidase obtained via an optimized multi-stage separation strategy for structural analysis and browning mechanism elucidation

An optimized multi-stage separation strategy was developed to purify lily bulb polyphenol oxidase (PPO) for revealing its molecular structure. The PPO was purified 14.64-fold with high specific activity of 153,900 U/mg via optimized conditions of phosphate buffer pH (6.5), solid-liquid ratio (1:3), PVPP content (2 %), extraction time (4 h), followed by 30 %–50 % ammonium sulfate, diethylaminoethyl ion-exchange chromatography (0.1 M NaCl), and size exclusion chromatography. The PPO was identified as a dimeric protein with molecular weight of 135 kDa, containing 58.79 % random coil, 20.78 % α-helix, 17.41 % β-folding, and 3.02 % β-corner. The three-dimensional structure via homology modeling suggested that active center CuA bound to His151, His172, and His181, CuB bound to His307, His311, and His341. Furthermore, molecular docking indicated that its Phe337 and Tyr312 residues were catalytic cavity gates of catechol and 4-methylcatechol, respectively. Therefore, this study successfully analyzed purified PPO structure and further provided a theoretical foundation for its browning mechanism.

Purification, cloning, expression, and biochemical characterization of a monofunctional catalase, KatP, from Pigmentiphaga sp. DL-8

Catalases are essential components of the cellular equipment used to cope with oxidative stress. The monofunctional catalase KatP was purified from Pigmentiphaga sp. using ammonium sulfate precipitation (ASP), diethylaminoethyl ion exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC). The purified catalase formed polymer with an estimated monomer molecular mass of 54kDa, which were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and zymogram analysis. KatP exhibited a specific catalytic activity of 73,000U/mg, which was higher than that of catalase-1 of Comamonas terrigena N3H (55,900U/mg). Seven short tryptic fragments of this catalase were obtained by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS/MS), and the gene, katP, was cloned by PCR amplification and overexpressed in Escherichia coli BL21 (DE3). Based on the complete amino acid sequence, KatP was identified as a clade 3 monofunctional catalase. The specific activities of recombinant KatP for hydrogen peroxide (690,000U/mg) increased 9-fold over that of the parent strain. The Km and Vmax of recombinant KatP were 9.48mM and 81.2mol/minmg, respectively. The optimal pH and temperature for KatP were 7.0 and 37°C, respectively, and the enzyme displayed abroad pH-stable range of 4.0–11.0. The enzyme was inhibited by Zn2+, Cu2+, Cr2+, and Mn2+, whereas Fe3+ and Mg2+ stimulated KatP enzymatic activity. Interestingly, the catalase activity of recombinant KatP displayed high stability under different temperature and pH conditions, suggesting that KatP is a potential candidate for the production of catalase.

Isolation, Partial Purification and Characterization of Texas Live Oak (<i>Quercus fusiformis</i>) Lectin

Lectins are carbohydrate-binding proteins with agglutination properties. There is a continuous interest in lectins due to their biological properties that can be exploited for medicinal and therapeutic purposes. The objective of this study was to isolate and characterize lectin activity in Texas Live Oak (Quercus fusiformis). More specifically, the study aimed to determine the lectin’s blood group specificity and pH stability, determine effects of seasonal variation, soil moisture and soil pH on lectin activity. The study also aimed to determine the presence of antifungal activity in Q. fusiformis extracts. Lectin activity was detected and compared via agglutination and protein assays. Protein partial purification was accomplished using diethylaminoethyl ion-exchange chromatography matrix. High Performance Liquid Chromatography (HPLC) was used to assess purity of the lectin. Results showed that Q. fusiformis extracts’ lectin activities are stable at a pH range of 5.2 - 9.2 but with a significant decrease in activity above pH 9.2. The lectin activity was significantly higher when assayed against sheep red blood cells as compared to other blood groups tested. Quercus fusiformis extract is devoid of antifungal activity against Aspergillus niger and Rhizopus stolonifer. The effects of seasonal variation, soil moisture and soil pH do not significantly correlate with lectin activity. Results from HPLC showed presence of three peaks indicating a partial purification of the Q. fusiformis lectin.

Expression, purification, and characterization of recombinant human hypoxia inducible factor 1α in E.coli

The Hypoxia-inducible factor 1 (HIF-1) heterodimeric transcription factor which is composed of a regulated α subunit and a constitutively expressed β subunit is a critical regulatory factor that orchestrate the cellular responses to hypoxia. It plays an important role in the pathology of many human diseases. The oxygen-sensing and transactivating functions of HIF-1 are contained within the α subunit, which is up-regulated in most human cancers. To study the function of HIF-1α more intensively, we designed a system to express and purify functional recombinant human HIF-1α (rhHIF-1α) protein from E. coli. The expressed His-tagged rhHIF-1α which forms inclusion bodies at 28°C was washed, dissolved and refolded firstly. Then the refolding His-tagged rhHIF-1α fusion protein was purified by diethylaminoethyl ion-exchange chromatography. After cleaved by thrombin, rhHIF-1α was thoroughly separated from tags by using Ni–NTA affinity chromatography, which was confirmed by SDS–PAGE and western blotting analysis. Thereafter, rhHIF-1α was further purified by using molecular sieve and its function was identified by electrophoretic mobility shift assay and the result indicated that the purified rhHIF-1α has DNA binding activity. The present expression and purification procedure enabled us to obtain biologically active rhHIF-1α from E. coli for in vitro function studies.

Partial purification and characterization of serum embryotrophic factor required for early postimplantation growth of rat embryos in culture.

Serum embryotrophic factor (SEF) required for the growth of cultured postimplantation rat embryos was partially purified from rat serum. Rabbit serum was used as a basal medium for the embryo culture, and embryotrophic activity was measured as embryonic protein increase. For partial purification of SEF, the rat serum globulin fraction obtained by ultracentrifugation and (NH4)2SO4 precipitation was fractionated by gel filtration, diethylaminoethyl ion-exchange chromatography, and hydroxyapatite chromatography. Partially purified SEF was characterized by stability tests and affinity chromatography. SEF was inactivated by heat, acid, dithiothreitol reduction, or trypsin digestion. SEF bound to concanavalin A but not to heparin. These results indicated that SEF was an acid-labile acidic glycoprotein with disulphide bonds and no affinity for heparin. The M(r) of SEF was estimated to be about 180 x 10(3) by gel filtration. The specific activity (U/g protein) was increased about 25-fold with 9.4% recovery by the partial purification, when 1 U of SEF was defined as the amount giving 50% embryonic protein increase. By polyacrylamide gel electrophoresis, a protein most likely to be SEF was identified as a heterodimer composed of subunits of M(r) 116 x 10(3) and 62 x 10(3) linked by disulphide bonds, and was shown to be contained in the medium at micromolar concentrations. SEF appeared to be distinct from known protein embryotrophic factors, growth factors, or cytokines.

Adrenocorticotropic hormone receptor-blocking immunoglobulins in serum from patients with Addison's disease: a reexamination.

The presence of antibodies inhibitory to ACTH-stimulated cortisol secretion was investigated in patients with Addison's disease. In preliminary experiments, immunoglobulin G (IgG) samples from patients with Addison's disease and control subjects were prepared using diethylaminoethyl-cellulose ion exchange chromatography. Twelve of 29 Addison's IgGs and 1 of 3 control IgGs inhibited ACTH-stimulated cortisol secretion in an in vitro guinea pig adrenal cell bioassay. All but 1 of the samples that inhibited ACTH-stimulated cortisol secretion also inhibited dibutyryl cAMP-stimulated steroidogenesis. IgGs purified by protein-G immunoaffinity were similarly screened for inhibition of ACTH-stimulated cortisol secretion; samples from 5 controls and 5 patients with Graves' disease were without effect, and IgGs from only 2 of 25 patients with Addison's disease decreased ACTH bioactivity (by < 12%). The effects of the IgGs on ACTH bioactivity in vitro were not concentration dependent. Our results suggest that Addison's disease is not caused by circulating antibodies to the ACTH receptor. The inhibitory effects seen with IgG prepared by diethylaminoethyl ion exchange chromatography were most likely attributable to the effects of non-IgG components in the preparations.

Intraparticle protein diffusion effect on gradient elution chromatography

The diffusion of proteins in adsorbent particles is an important step in gradient elution processes used for protein purification. A mathematical model, which takes into account the combined effects of axial dispersion, film mass-transfer resistance, intraparticle diffusion and adsorption equilibrium, was used to simulate the gradient elution process in a diethylaminoethyl ion-exchange chromatography. The effects of three important factors: intraparticle diffusivity, adsorbent particle diameter, and linear gradient slope on the separation of β-lactoglobulin A and β-lactoglobulin B were investigated individually. The restricted diffusion correlation was used to estimate protein intraparticle diffusivities in polymer matrices using the molecular weight of the protein and the polymer concentration (Boyer, P. M. and Hsu, J. T. AIChE J. 1992, 38, 259). The combined effects of these three factors on the resolution of β-lactoglobulin A and β-lactoglobulin B separation were also studied. A general strategy for design and optimization of gradient elution process for biochemical separations was suggested based on our investigation.

Purification and characterization of rabbit pancreas protein kinase C.

Protein kinase C was purified 6,900-fold from rabbit pancreas with a total yield of 15% by a procedure involving ammonium sulfate fractionation, diethyl aminoethyl ion exchange chromatography, hydroxylapatite chromatography, and finally protamine-agarose affinity chromatography. After these purification steps the protein kinase C preparation contained two major protein bands as judged by silver staining after SDS-polyacrylamide gel electrophoresis: 80 and 69-kDa bands. Monoclonal antibodies directed against bovine brain protein kinase C (alpha- and beta-subtype) recognized only the 80-kDa band. On the other hand, both the 80 and 69-kDa proteins were recognized by a polyclonal monospecific antibody directed against rat brain protein kinase C. Analysis of rabbit pancreas protein kinase C subtypes by means of hydroxylapatite chromatography showed the presence of the III (alpha) subtype as the major subtype. The enzyme depended absolutely on the presence of both phosphatidylserine and Ca2+ for its activity, with apparent Ka values of 3.1 micrograms/ml and 247 microM for phosphatidylserine and Ca2+, respectively. When dioctanoylglycerol or the phorbol ester 12-O-tetradecanoyl-phorbol 13 acetate (TPA) was present, the Ka value for Ca2+ decreased to 10 and 18 microM, respectively. In the presence of the phorbol ester, pancreatic protein kinase C could be activated without added Ca2+. The enzyme also required Mg2+ for its activity. The Ka value was 3.6 mM and maximal activity was reached at 10 mM Mg2+. Pancreatic protein kinase C activity showed a broad pH dependence, with optimal activity at pH 6.75. The Km value for ATP and for histone-H1 was 8.5 microM and 20.4 micrograms/ml, respectively. The present study shows that the kinetic properties of protein kinase C purified from rabbit pancreas closely resemble those found in other tissues.

Immunochemical Detection of Predation on Ciliate Protists by Larvae of the Northern Anchovy (Engraulis mordax).

New approaches are needed for investigating planktonic prey-predator interactions in situ, free from enclosures and long term incubations. Lengthy incubations can lead to several artifacts, including selective mortality of planktonic organisms (1) lysis of some taxa upon fixation (2), alteration of prey-predator encounter rates through perturbations of the turbulent flow field (3) and modification of natural search and avoidance behaviors of predators and prey (4). Microscopic analysis of the gut contents of predators is a feasible alternative only if prey leave digestion-resistant hard parts. Here we describe a different approach involving immunochemical methods. We report the development and first application of an immunoassay that permits detection of predation on soft-bodied, nonloricate ciliates (Strombidium sp.). Polyclonal antibodies raised against Strombidium sp. recognize both intact ciliates and partially assimilated ciliate antigens occurring in a predator’s gut. We demonstrate, by both immunochemical and conventional methods, unequivocal predation by first-feeding larvae of the northern anchovy (Engraulis mordax) on nonloricate ciliates. The intensity of the immunochemical reaction, quantified by enzymelinked dot blots and reflection densitometry, is proportional to prey density and to the predator’s ingestion rate. Polyclonal antisera were produced in New Zealand white rabbits (5) against Strombidium sp. cultured on a diet of the bacterium Vibrio natriegens (6). Immunoglobulin G (anti-Strombidium IgG) was isolated from the antiserum by precipitation in 45% (w/v) ammonium sulfate, desalted by gel filtration, then fractionated by diethylaminoethyl ion exchange chromatography. Detailed

Enzymatic Detoxification of HC-toxin, the Host-Selective Cyclic Peptide from Cochliobolus carbonum

Resistance to the fungal plant pathogen Cochliobolus carbonum race 1 and to its host-selective toxin, HC-toxin, is determined by Hm, a single dominant gene in the host plant maize, (Zea mays L). Radiolabeled HC-toxin of specific activity 70 milliCuries per millimole, prepared by feeding tritiated d,l-alanine to the fungus, was used to study its fate in maize leaf tissues. HC-toxin was converted by resistant leaf segments to a single compound, identified by mass spectrometry and nuclear magnetic resonance as the 8-hydroxy derivative of HC-toxin formed by reduction of the 8-keto group of 2-amino-9, 10-epoxy-8-oxo-decanoic acid, one of the amino acids in HC-toxin. Reduction of HC-toxin occurred in cell-free preparations from etiolated (Hm/hm) maize shoots, and the activity was sensitive to heat and proteolytic digestion, dependent on NADPH, and inhibited by p-hydroxymercuribenzoate and disulfiram. The enzyme (from the Hm/hm genotype) was partially purified by ammonium sulfate precipitation and diethylaminoethyl-ion exchange chromatography. By gel filtration chromatography, the enzyme had a molecular weight of 42,000. NADH was approximately 30% as effective as NADPH as a hydride donor, and flavin-containing cofactors had no effect on activity. When HC-toxin was introduced to maize leaf segments through the transpiration stream, leaf segments from both resistant and susceptible maize inactivated toxin equally well over a time-course of 9 hours. Although these data suggest no relationship between toxin metabolism and host selectivity, we discuss findings in apparent conflict with the current data and describe why the relationship between enzymatic reduction of HC-toxin and Hm remains unresolved.

Isolation of β-Lactogobulin and α-Lactalbumin by Gel Filtration Using Sephacryl S-200 and Purification by Diethylaminoethyl Ion-Exchange Chromatography

β-Lactoglobulin dimer (408mg) was easily and gently separated from neutralized acid whey (200ml) by gel filtration as the fourth peak eluted from Sephacryl S-200. This β-lactoglobulin was separated into A (63%) and B (37%) variants by DEAE ion-exchange chromatography ants each showed single bands of similar mobility on SDS-PAGE and double bands with like mobilities on SDS-urea-PAGE however, the variants showed different mobilities on native PAGE.α-Lactalbumin (98mg) was also easily and gently separated by the gel filtration as the fifth peak (134mg protein). Other proteins recovered in P5 fraction were trace amounts of β-lactoglobulin monomer and an unidentified albumin (14,000Da). After purification by Aschaffenburg and Drewry's method, β-lactoglobulin monomer was absent from α-lactalbumin fraction; however, the unidentified albumin remained.

Purification and Characterization of a Bovine Cerebral Cortex Cell Surface Sialoglycopeptide that Inhibits Cell Proliferation and Metabolism

A sialoglycopeptide from bovine cerebral cortex cells was purified to apparent homogeneity by a procedure that included chloroform/methanol extraction, diethylaminoethyl ion exchange chromatography, wheat germ agglutinin affinity chromatography, size-exclusion HPLC, and hydrophobic interaction chromatography. The cell surface inhibitor had a molecular weight of approximately 18,000, no subunit composition was detectable on reduction and polyacrylamide gel electrophoresis analysis, and the glycopeptide apparently contained sialic acid, as illustrated by its ability to bind to Limulus polyhemus lectin. Deglycosylation of the molecule, however, did not reduce its protein synthesis inhibitory activity. As little as 20 ng of the sialoglycopeptide was capable of inhibiting protein synthesis in a wide variety of fibroblast cell lines but not in transformed cells. Mice immunized with the sialoglycopeptide produced antibodies that, when bound to protein A-agarose gel, removed the inhibitory activity from solution. The antibodies were used to identify a single isoelectric focused band and to establish the pI of 3.0 for the molecule.

Characterization of the Glycoprotein Antigens Which Mediate the Schistosoma japonicum Circumoval Precipitin Reaction

The circumoval precipitin test is a serological test used for diagnosis of schistosomiasis japonica. Soluble egg antigens of Schistosoma japonicum block the formation of the circumoval precipitin by serum from infected humans. Consequently, circumoval precipitin inhibition was used to monitor purification of the soluble egg antigens of S. japonicum. Crude egg antigens were separated into protein and glycoprotein fractions by lectin chromatography on concanavalin A Sepharose. The glycoprotein fraction produced two intense precipitin lines upon immunodiffusion analysis with human chronic infection sera. The protein fraction produced two faint precipitin lines which did not cross-react with those of the glycoprotein fraction. The glycoprotein fraction contained 90% of the circumoval precipitin inhibitory activity. Isoelectric focusing of (125)I-labeled concanavalin A Sepharose fractions revealed at least four groups of potential S. japonicum antigens, termed JAG I, II, and III, and a JAG IV complex. These had isoelectric points ranging from 3.2 to 6.7. In these respects, the S. japonicum egg antigen glycoproteins are similar to those of Schistosoma mansoni. The glycoproteins were further separated by diethylaminoethyl ion-exchange chromatography. On immunodiffusion analysis it was found that one of the strong Ouchterlony precipitin lines was associated with glycoproteins that did not adsorb to diethyl-aminoethyl columns, whereas the second Ouchterlony precipitin was heterodisperse, being present in the first, second, and third of the four peaks eluted from the diethylaminoethyl column. Immunoelectrophoresis of the diethylaminoethyl fractions demonstrated that the antigen present in highest concentration in soluble egg antigen glycoproteins, JAG II, was extremely heterodisperse in its behavior on diethylaminoethyl columns. This is unlike the S. mansoni antigens which can be easily separated by diethylaminoethyl ion-exchange chromatography.

Concerning chemistry and testing of bence-jones proteins

The amino acid composition and terminal amino acids of two typical individual specimens of Bence-Jones proteins were determined. The amino acid composition of the specimens was very similar, and it was noteworthy with its high hydroxyamino acid content. Subfractionation of one of the specimens was accomplished by the diethylaminoethyl ion exchange chromatography. It was also found that the specimens contained half-cystine, in addition to aspartic acid, as the N-terminal group. The halfcystine, however, was present only in one of the subfractions, while the others were devoid of it. The proteins showed the characteristic propanol flocculation behavior which can be used for their testing in urine for diagnostic purposes.