Binding of human interferons to immobolized Cibacron Blue F3GA: The nature of molecular interaction

Blue Dextran (Cibacron Blue F3GA-dextran) was immobilized on cyanogen bromide activated agarose and used as a ligand for human fibroblast and leukocyte interferons in a solvent of phosphate-buffered (pH 7.4), physiological saline (0.15 M NaCl). Fibroblast interferon binds completely and is not displaced from the column by an increase in ionic strength of the solvent (1.0 M NaCl); it can be, however, recovered with ethylene glycol, indicating the hydrophobic nature of interaction. Leukocyte interferon also binds to Blue Dextran-agarose but it can be recovered simply by an increase in the ionic strength of the solvent, indicating primarily the electrostatic nature of binding. Attempts to displace both interferons selectively with nucleosides and aromatic amino acids were unsuccessful. When Cibacron Blue F3GA is immobilized directly to agarose matrix or via molecular arm, the strength of binding of fibroblast interfern is significantly decreased, although ethylene glycol is still required for its displacement from the column. Leukocyte interferon, by contrast, does not bind at all under the same solvent conditions; it does bind when the pH value of the solvent is in the range 3-5 i.e., below its isoelectric point. Human fibroblast interferon binds completely to: aminobenzene, aminonaphthalene, and aminoanthracene, all immobilized on agarose, and it can be recovered with ethylene glycol. In contrast, human leukocyte interferon does not bind to benzene-agarose; it is retarded on naphthalene-agarose and completely retained on an anthracene-agarose column. All data point to a higher intrinsic hydrophobicity of human fibroblast interferon vis-á-vis human leukocyte interferon. Selective binding of human fibroblast interferon of Cibacron Blue F3GA-agarose results in a significant purification, about 800-fold.     

Effects of thiols on prostaglandin synthesis in bovine bladder epithelium

Immunoaffinity chromatography was shown to be the method achieving the most complete elimination of antigenic admixtures from leukocyte interferon preparations without the loss of the preparation activity. An affinity sorbent has been developed on the basis of covalently linked polyvinyl alcohol (PVA). The immobilization of the antigen-specific rabbit globulin in the preparation of the sorbent is achieved by reaction of protein amino groups with the activated matrix. The proposed sorbent achieved the elimination of the antigenic admixtures from interferon preparations as effectively as those prepared on the basis of sepharose 4B, the productivity of the purification process being at least 5 times higher. The proposed sorbent is stable at the limit values of rH, is not destroyed by detergents, is sterilized in the process of preparation. Owing to the strong linkage of the immobilized immunoglobulin with the PVA-carrier, immunoaffinity chromatography on this sorbent does not involve contamination of the preparations with rabbit globulin allergenic for man. The combination of a large pore structure, wetting ability, stiffness, mechanical and chemical stability allows the proposed sorbent to be recommended for use in modern large-scale biotechnological production.     

Computer simulation of expansions of DNA triplet repeats in the fragile X syndrome and Huntington''s disease

The use of high capacity micron-sized non-porous magnetic metal chelator adsorbents for the direct recovery of a recombinant metal-binding protein from crude liquors is described. Selectivity and interaction strength of magnetic chelator particles were assessed using a set of native proteins with known behaviour towards commercially available immobilised metal chelate adsorbents. Particles charged with Cu2+ were highly effective in recovering a recombinant histidine-tailed T4 lysozyme fusion protein directly from crude E. coli extracts in a single step. Levels of recovery and purity were high and compared favourably with those achieved by chromatography of pre-clarified extracts on Cu(2+)-IDA Sepharose. The magnetic approach offers advantages such as the avoidance of clarification to prevent fouling of chromatography columns, steps that become especially significant at large scale. By detailed characterisation of the magnetic chelators the practical use of tailed T4 lysozyme for repeated production of periplasmic products is a realistic prospect.     

Interferon-alpha 2 produced by normal human leukocytes is predominantly interferon-alpha 2b

Peripheral blood leukocytes, isolated from the buffy coats of greater than 10,700 normal healthy donors, were induced with Sendai virus to produce biologically active interferon alpha (IFN-alpha). The IFN-alpha was purified to near homogeneity by immunoaffinity chromatography, followed by size-exclusion chromatography. The resultant product, IFN-alpha n3, is reproducibly > or = 98% pure (to be reported elsewhere). The different IFN-alpha proteins in IFN-alpha n3 were separated by reverse-phase high performance liquid chromatography (RP-HPLC) and the identity of the IFN-alpha 2 isolated by HPLC was determined by amino-terminal sequencing. IFN-alpha 2 was found to migrate as two closely eluting peaks on RP-HPLC, and they have been designated as peaks 1.1 and 1.2. Distinction among the three possible variants of IFN-alpha 2, i.e., IFN-alpha 2a, IFN-alpha 2b, and IFN-alpha 2c, was determined by amino-terminal sequencing of the first 35 amino acids in peaks 1.1 and 1.2. Protein sequence data showed that the discriminating amino acids found at positions 23 and 34 are Arg and His, respectively. The presence of Arg and not Lys at amino acid position 23 and His at amino acid position 34 argues that IFN-alpha 2b is the major component in the Sendai virus-induced leukocyte IFN-alpha 2 and that IFN-alpha 2a is not present. These findings were verified by subjecting RP-HPLC peaks 1.1 and 1.2 to CNBr cleavage, followed by separation of the fragments by RP-HPLC and sequencing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbon Fiber Adsorption Using Quantitative Structure-Activity Relationship

Adsorption capacities of adsorbents are necessary for selecting and designing adsorption systems for separation and removal processes, such as air quality control devices, because they are indicators of service life. This paper describes the use of the Dubinin-Radushkevich (DR) equation and the quantitative structure-activated relationship to predict the equilibrium adsorption of select organic vapor by activated carbon fiber (ACF) adsorbents. The DR isotherm parameter, k, depends on the adsorbate as well as the adsorbent, and the prediction for k can be obtained indirectly from the affinity coefficient. A correlation is developed to compute the affinity coefficient from the modified, first-order, valence molecular connectivity index. This method provides an improved way to predict equilibrium adsorption capacities for select volatile organic compound adsorbates and activated carbon fiber adsorbents.     

The separation and characterization of the methylumbelliferyl beta-galactosidases of human liver

1. A previously uncharacterized form of human liver acid beta-galactosidase (EC 3.2.1.23), possibly a dimer of molecular weight 160 000, was resolved by gel filtration. It has the same ability to hydrolyse GM1 ganglioside as the two other acid beta-galactosidase forms. 2. The low-molecular-weight forms of acid beta-galactosidase undergo salt-dependent aggregation. 3. The high-molecular-weight component may consist of the low-molecular-weight forms bound to membrane fragments. It can be converted completely into a mixture of these forms. 4. The neutral beta-galactosidase activity can be resolved into two forms by DEAE-cellulose chromatography. They differ in their response to Cl-ions. 5. A new nomenclature is suggested for the six beta-galactosidases so far found in human liver. 6. The enzymic constituents of the beta-galactosidase bands resolved by electrophoresis were re-examined. The A band contains three components. A two-dimensional electrophoretic procedure for resolving the A band is described. 7. The effect of neuraminidase treatment on the behaviour of beta-galactosidases in various separation systems is examined.     

Estimation of the partitioning characteristics of drugs: a comparison of a large and diverse drug series utilizing chromatographic and electrophoretic methodology

1-Octanol-water log P values for a large number of standards and bioactive molecules have been correlated to the logarithm of the corresponding capacity factors determined by reversed-phase high-performance liquid chromatography, using a novel dynamically coated phase, containing phosphatidylcholine. Similarly a correlation was also obtained for log P and capacity factors determined by micellar electrokinetic capillary chromatography (MECC), involving the use of phosphatidylcholine--bile acid mixed micelles in the separation buffer. Statistical analysis of data obtained via both methods has shown that either method will give reliable log P predictions, although MECC is generally more useful for neutral and basic compounds. It is recommended that, as both methods can easily be set up in an analytical laboratory, their combined use provides rapid methodology for the confident estimation of hydrophobicity, as measured by log P for the widest diversity of chemical structures.     

Structural characterization of synthetic model peptides of the DNA-binding cI434 repressor by electrospray ionization and fast atom bombardment mass spectrometry

The structural characterization of two synthetic model peptides of the cI434 repressor is described. Unequivocal determination of the structure was achieved by means of electrospray ionization mass spectrometry of the intact peptides and by fast atom bombardment mass spectrometric identification of complementary peptide fragments obtained by tryptic and chymotrypic digestion and partial separation by reversed-phase high-performance liquid chromatography. The results show the potential of this approach for characterizing synthetic peptides of relatively high molecular weight.     

Heterogeneity of carbohydrate chains of acidic bronchial mucin isolated from the spatum of two subjects with chronic bronchitis

Acidic glycoproteins having blood group H activity were isolated from the sputum of two patients suffering from chronic bronchitis by reduction of the fibrillar mucus, chromatography on ECTEOLA-cellulose and gel filtration on Sepharose 4-B. These glycoproteins were degraded with alkaline borohydride and the degradation products were fractionated by chromatography on ionexchange resins and by gel filtration. The carbohydrate chains have a wide heterogeneity with regard to acidity and molecular size. Therefore carbohydrate chain heterogeneity which was already observed for bronchial glycoproteins isolated from a patient suffering from cystic fibrosis is not specific for cystic fibrosis.     

Enhancement of sample loadings for the analysis of oligosaccharides isolated from Pseudomonas aeruginosa using transient isotachophoresis and capillary zone electrophoresis - electrospray - mass spectrometry

The analysis of underivatized core oligosaccharides arising from mild acid hydrolysis of lipopolysaccharides from Pseudomonas aeruginosa serotype 05 was achieved using a transient isotachophoretic preconcentration method coupled to capillary zone electrophoresis-electrospray-mass spectrometry (tCITP-CZE-ES-MS). The combination of a tCITP preconcentration step provided a 10- to 50-fold enhancement of sample loading and a corresponding improvement in sensitivity compared to the conventional zone electrophoresis format. Electrophoretic conditions, enabling the separation of these anionic analytes, were developed to determine possible sites of heterogeneity on either the core or the O-chain structures. The tCITP-CZE-ES-MS technique provided unparalleled resolution of the different core glycoforms and oligosaccharides obtained from the acid cleavage of the native endotoxins whether isolated following conventional gel permeation chromatography or obtained from direct hydrolysis of the bacterial isolates. These investigations also highlighted the highly phosphorylated nature of these complex cell membrane components, where the heptose residues of the core oligosaccharide can bear up to six phosphate groups.     

Glucocorticoid binding in the chicken liver cytosol. Characterization of five macromolecular binding components

The heterogeneity of the glucocorticoid binding in the chicken liver cytosol, previously suggested by the results obtained with crude preparations, was confirmed using different techniques such as stepwise ammonium sulfate precipitation, hydroxyapatite chromatography and gel filtration on Sephadex G-200. The latter method permitted the separation of five glucocorticoid binding macromolecules respectively named binders S1, S2, S3, S4 and S5, according to their decreasing apparent molecular size upon gel filtration. Apparent molecular weight, binding affinity, capacity and specificity of these five moieties were examined. In addition, S-aryl-transferase activity using glutathione as co-substrate was studied and found to coincide mostly with the fractions containing binder S4, which might represent an avian liver ligandin.     

Chemical composition of the capsule material of Staphylococcus aureus strain 1193/74

The capsule material of Staphylococcus aureus strain 1193/74 could be separated by precipitation with trichloroacetic acid and ethanol as well as by chromatography on DEAE-cellulose into 13 fractions. All fractions contained saccharides and uronic acids as well as amino acids and appeared in their qualitative composition rather similar. However, in quantitative composition and in chromatographic behaviour a rather high degree of heterogeneity could be observed. No clear cut separation of protein and polysaccharide material could be achieved in any fraction. It is supposed, therefore, that the capsule material does not represent merely an acidic polysaccharide, but contains certain amounts of amino acids or peptides varying in a rather wide range.     

Passive immunity in the lamb. Effects of a second feed of colostrum on antibody absorption from the first feed

Bone marrow cells from suddenly dying people can produce interferon in response to incoulation of an inducer. The interferon obtained in bone marrow cells did not differ by its properties from the reference preparation. the lots of bone marrow interferon which had been prepared were not inferior in the antiviral activity to leukocyte interferon or were even superior to it. It is suggested that bone marrow cells be used for preparation of human interferon.     

Congenital neutropenia: an intrinsic cell defect demonstrated by electron microscopy of soft agar colonies

In an attempt to define the molecular events involved in induction of interferon, various parameters of chick cells infected with human adenovirus type 5 were analysed. It was shown by digestion with various proteolytic enzymes and by disruption of the purified virus that induction of interferon requires the interaction of infectious virus with the chick cells. Analysis of adenovirus-infected chick cells by immunological and biochemical techniques indicated that most of the cells produce some virus-specific components, and that temperature sensitive mutants which fail to induce interferon at the restrictive temperature fail to synthesize late components at that temperature. However, since it has been shown that interferon can be induced in the absence of DNA synthesis, these studies conclude that interferon induction results from an early interaction between virus (or virus product) and chick cells and moreover that this interaction is also necessary for the synthesis of virus DNA in this system.     

Effects of piracetam on the performance of rats in a delayed match-to-position task

The growing use of antibody-based separation methods has paralleled the expansion of immunochemical detection methods in moving beyond the clinical diagnostic field to applications in environmental monitoring. In recent years high-performance immunoaffinity chromatography, which began as a separation technique in biochemical and clinical research, has been adapted for separating and quantifying environmental pollutants. Bioaffinity offers a selective biological basis for separation that can be incorporated into a modular analytical process for more efficient environmental analysis. The use of immunoaffinity chromatography for separation complements the use of immunoassay for detection. A widely used immunochemical detection method for environmental analyses is enzyme immunoassay. The objective of this paper is to review the status of bioaffinity-based analytical procedures for environmental applications and human exposure assessment studies. Environmental methods based on bioaffinity range from mature immunoassays to emerging techniques such as immunosensors and immunoaffinity chromatography procedures for small molecules.     

Effect of human leukocyte interferon on hepatitis B virus infection in patients with chronic active hepatitis

Four patients with chronic hepatitis B infection and chronic active hepatitis were treated with human leukocyte interferon. Three of them had consistently elevated levels of circulating Dane-particle markers, including Dane-particle-associated DNA polymerase activity, hepatitis B core antigen and Dane-particle-associated DNA. Parenteral interferon administration at a dosage between 6.0 X 10(3) and 17 X 10(4) U per kilogram per day was associated with a rapid and reproducible fall in all Dane-particle markers in the three patients. The suppressive effect was transient when the interferon was given for 10 days or less but appeared to be more permanent when administration was prolonged for a month or more. In addition, long-term interferon therapy was associated with a marked fall in hepatitis B surface antigen in two of three patients and a disappearance of e antigen in two of two patients. Interferon may be useful in limiting carrier infectivity or eradicating chronic infection.     

Capillary liquid chromatography/electrospray mass spectrometry for the separation and detection of catechins in green tea and human plasma

The separation and detection of biologically active green tea catechins has been accomplished using capillary liquid chromatography/electrospray mass spectrometry (cLC/ESI-MS). Microscale determination (approximately 20 ng) of all six catechins in a green tea infusion, and the most extensively studied catechin, (-)epigallocatechin gallate (EGCG), in human plasma is demonstrated by cLC/ESI-MS with selected ion monitoring of protonated molecular ions. The overall quality of the analysis is shown to be dependent on the use of a capillary column with a deactivated, monomeric C18 stationary phase. The high chromatographic separation efficiency of this packed-capillary column, combined with the high sensitivity and selectivity afforded by the mass spectrometer as detector, provide a reliable approach to the analysis of picomolar quantities of these interesting compounds in complex matrices.     

The use of microemulsion electrokinetic chromatography in pharmaceutical analysis

The use of a single set of microemulsion electrokinetic chromatography (MEEKC) separation conditions has been assessed for its applicability in the analysis of a range of pharmaceutical compounds. Particular emphasis was placed on neutral or very hydrophobic compounds, which can be difficult to analyse by conventional capillary electrophoresis. The microemulsion employed for the majority of separations consisted of 0.81% w/w octane, 6.61% w/w 1-butanol, 3.31% w/w sodium dodecyl sulphate and 89.27% w/w 10 mM sodium tetraborate buffer. Good separations of methyl, ethyl, butyl and propyl hydroxybenzoates, and a range of ionic and neutral water soluble and insoluble compounds was achieved using a single set of separation conditions. A number of novel applications of MEEKC were developed included the simultaneous determination of the active components and preservatives in liquid formulation and determination of drug related impurities. Improved performance was obtained through use of internal standards and preparation of the samples dissolved in the microemulsion solution. Validation aspects such as linearity, repeatability, accuracy, injection precision and sensitivity were successfully assessed.     

Capillary affinity chromatography and affinity capillary electrophoresis of heparin binding proteins

A new approach for separation, capillary affinity chromatography, is introduced for studying the interaction of heparin with antithrombin III and secretory leukocyte proteinase inhibitor. Heparin is covalently immobilized on the surface of an etched capillary through a silane spacer. The proteins are injected into the heparinized capillary, bound to the heparin, washed with buffer, eluted with sodium chloride in the same buffer using a pressure injection mode and eluting protein detected by absorbance. The resulting affinity separation is similar to that obtained from traditional affinity chromatography. The quantity of loaded protein in capillary affinity chromatography is at the nanogram level, offering an improvement over the milligram levels required for standard affinity chromatographic methods.