Preparation of a stable folate-sepharose complex for affinity chromatography.

A stable folic acid affinity gel has been developed for the purification of nanograms of protein that bind folic acid or its derivatives. The affinity gel was prepared by first coupling folic acid covalently to bovine serum albumin, followed by covalent coupling of the albumin to p-benzoquinone-activated Sepharose. After the albumin-folic acid complex was formed, it was treated with charcoal to remove ionically bound folate which would otherwise elute from the gel and decrease the recovery of the binding protein. The p-benzoquinone activation resulted in a more stable binding of the albumin to the Sepharose.     

Human alpha-chain globin messenger: prediction of a nucleotide sequence

The presence in human serum of inhibitory activity to rat liver insulin specific protease has been detected in an alpha₁ globulin preparation (Cohn Fraction IV₁). Separation into four components and partial purification (40 to 107 fold) has been achieved by heat denaturation of non-active protein, Sephadex G-100 gel filtration and ion-exchange chromatography upon QAE Sephadex. Each of the inhibitors was found to be competitive in nature. The molecular weight of the inhibitors is between 4,000–7,000 and the activity is destroyed for the most part by chymotrypsin.     

Aminopropyl silica gel as a solid support for preparation of glycolipid immunoadsorbent and purification of antibodies.

Aminopropyl silica gel was prepared from porous silica gel and was used as a solid support for immunoadsorbent in the purification of anti-glycolipid antibodies. For neutral glycosphingolipids, a carboxyl function was generated by oxidation of the olefinic double bond of the sphingosine moiety, whereas for gangliosides the carboxyl group of sialic acid was used to couple with aminopropyl silica gel in the presence of a carbodiimide. These compounds were used for purifying anti-glycolipid antibodies from serum of immunized rabbits. The antibodies bound to the su-strate were released by 2 M potassium thiocyanate and their immunological properties were studied. Aminopropyl silica gel may be preferred over conventional organic solid supports for the following reasons: 1) faster flow rate; 2) higher capacity; 3) easier handling; 4) more economical; and 5) lower susceptibility to microbial attack.     

Characterization of a lymphokine produced by human T cells which inhibits collagen synthesis

Human lymphocytes, isolated from peripheral blood, were cultured for 48 hr in a defined medium containing 10 mg/ml bovine serum albumin and phytohemagglutinin. A lymphokine which inhibits collagen synthesis by cultured human dermal fibroblasts was purified from the lymphocyte incubation medium by successive steps of ammonium sulfate precipitation, gel filtration chromatography, and isoelectric focusing. Good recovery of this collagen synthesis inhibitory factor (CSIF) was obtained and a factor with an approximate molecular weight of 55,000 and a pI of 6.2 was isolated. The purification of the factor should permit further studies on its mechanism of action.     

A new method of preparation of pyocyanin and demonstration of an unusual bacterial sensitivity.

Pyocyanin was prepared in 60% yield from phenazine methoxysulfate by a photooxidation procedure and purification by silica gel chromatography. Monitoring was performed by thin-layer chromatography. Approximately 50% of clinical Pseudomonas aeruginosa isolates were found to produce pyocyanin at 37°C. Among Proteus strains, P. morganii strains were sensitive to concentrations of pyocyanin 16 to 64 times lower than concentrations that inhibited the growth of P. mirabilis and P. vulgaris strains.     

The synthesis of 4-methylumbelliferyl alpha-ketoside of N-acetylneuraminic acid and its use in a fluorometric assay for neuraminidase

4-Methylumbelliferyl α-ketoside of N-acetylneuraminic acid was synthesized by reacting the sodium salt of 4-methylumbelliferone with the 2-chloro-2-deoxy derivative of peracetylated methyl N-acetylneuraminate, followed by preparative silica gel chromatography, deblocking, and purification by gel filtration on Sephadex G-25. The final product was isolated as either the sodium or ammonium salt, and its suitability as a substrate for neuraminidase was evaluated. The optimal pH values for various neuraminidases were 5.6 in acetate buffer (Arthrobacter ureafaciens), 5.0–5.1 in acetate buffer (Clostridium perfringens), and 4.4 in phosphate-citrate buffer (human fibroblasts). K_m values for these enzymes at the optimal pH were 6 × 10^?4m (Arthrobacter), 1 × 10^?4m (Clostridium), and 3 × 10^?4m (human fibroblasts).     

The simultaneous determination of the products of estrogen 2- and 4-hydroxylase action; the use of high-performance liquid chromatography with electrochemical detection

Both trace-labeled and high-specific activity ¹²⁵I-labeled derivatives of hexadecapeptide gastrin (G) were prepared by reaction with the iodinated form of the imidoester, methyl p-hydroxybenzimidate. Reaction conditions for preparation of trace-labeled iodinated imidoester gastrin (IIE-G) were: excess imidoester to G (IIE:G, 20:1), pH 9.2, and a reaction time of 24 h. Following purification by gel filtration and silica gel chromatography, an IIE-G component was isolated which appeared homogeneous on thin-layer chromatography and retained the same biological and immunological properties as unmodified G. Somewhat different conditions were necessary to prepare high specific activity iodinated imidoester gastrin (IIE¹-G). These included reducing the volume (20 μl) and pH (7.5) at which the imidoester was iodinated and adjusting the concentrations of reactants to the same molar amounts as 5 mCi of carrier-free ¹²⁵I. Sufficient amounts of IIE¹-G were obtained by reversing the ratio of G and IIE¹ and reacting with a G excess (GIIE¹, 10:1). The purified IIE¹-G had a specific activity exceeding 1500 μCi/nmol and was used to establish a specific and sensitive radioimmunoassay for gastrin.     

Column chromatography on polyethylenimine-silica: rapid resolution of nucleotides and proteins with short columns and low pressures

Silica gel (100–200 mesh) can be coated with a stable layer of crosslinked polyethylenimine (PEI). The resulting material is useful in column chromatography for quantitative separation of adenine nucleotides. For example, 3′:5′-cyclic AMP can be separated from a mixture containing AMP, ADP, and ATP. A mixture of adenosine, AMP, ADP, and ATP can be resolved with quantitative recovery of components. Convenient separation of cytochrome c from albumin illustrates the applicability of this system to protein purification. PEI-Xama silica gel and PEI-glutaraldehyde silica gel have ion-exchange capacities of 0.27 and 0.21 meq/g, respectively. The materials are dimensionally rigid and chemically stable except in alkaline solutions (>pH 10) for prolonged periods.     

Purification of Growth-Promoting Peptides and Proteins,and of Histones,by High Pressure Silica gel Chromatography

A rapid method for the purification of histones and a variety of growth-promoting proteins and peptides by chromatography on silica gel has been developed. The isolation of the growth-promoting components of serum has been hampered by excessive losses associated with the use of water-based purification methods. The solubility of many growth-promoting serum proteins in acidic methanol-H₂O solutions (eg. insulin, albumin, the somatomedins) provides a basis for purification on high-pressure silica gel columns, while peptides and histones can be purified in similar solvents. After column chromatography, the solvent is removed by flash-evaporation, or the protein may be precipitated directly from the solvent by neutralization of the pH and the addition of ethanol. The retention of biological activity (eg. somatomedin-C binding to insulin receptors and cell-growth stimulation) and recovery are excellent.     

Characterization of a small apolar anion binding site of human serum albumin.

Small unbranehed fatty acid anions inhibit the fast reaction between p-nitrophenylacetate and human serum albumin. Plots of reactivity versus fatty acid anion-albumin ratios resemble simple binding isotherms from which corresponding dissociation constants have been calculated. For the homologous fatty acid anions, butyrate through decanoate, dissociation constants decrease from 3.2 × 10^?4 to 1 × 10^?7m, respectively, by uniform increments per methylene group according to the relationship ?ΔG °(kcal) = 0.804_n + 2.30, where n is the number of constituent methylene groups. Small fatty acid anions thus appear to interact primarily with a single, relatively uniform apolar binding site with a capacity sufficient for nine methylene groups. Fatty acid anions larger than decanoate interact significantly with other sites and do not obey the same relationship. The reactivity of diluted human serum with p-nitrophenylacetate was found to be one-third to one-half of that expected for its content of serum albumin, but as in vitro, it could be completely inhibited by small amounts of decanoate.     

Adsorption of human serum proteins onto TREN-agarose: Purification of human IgG by negative chromatography

Tris(2-aminoethyl)amine (TREN) – a chelating agent used in IMAC – immobilized onto agarose gel was evaluated for the purification of IgG from human serum by negative chromatography. A one-step purification process allowed the recovery of 73.3% of the loaded IgG in the nonretained fractions with purity of 90–95% (based on total protein concentration and nephelometric analysis of albumin, transferrin, and immunoglobulins A, G, and M). The binding capacity was relatively high (66.63 mg of human serum protein/mL). These results suggest that this negative chromatography is a potential technique for purification of IgG from human serum.     

Entrapment of proteins in glycogen-capped and hydrazide-activated supports

A method is described for the entrapment of proteins in hydrazide-activated supports using oxidized glycogen as a capping agent. This approach is demonstrated using human serum albumin (HSA) as a model binding agent. After optimization of this method, a protein content of 43 (±1) mg of HSA/g support was obtained for porous silica. The entrapped HSA supports could retain a low-mass drug (S-warfarin) and had activities and equilibrium constants comparable to those for soluble HSA. It was also found that this approach could be used with other proteins and binding agents that had masses between 5.8 and 150 kDa.     

Use of porous pyrolytic carbon for analytical and microscale high-performance liquid chromatographic bioseparations

Porous carbonaceous adsorbent was prepared by carbonization of saccharose in silica gel pores followed by leeching out of the silica matrix. The product of pyrolysis was then deactivated by hydrogenation. The resulting adsorbent shows intermediate sorption properties between those of the highly polar pyrolytic glassy carbon and the hydrophobic graphitized carbon. The microparticulate mesoporous carbon was examined for its use in capillary HPLC separations. The separation of selected stereoisomers in a 320 μm I.D. capillary column packed with the porous carbon particles is described and discussed. Additionaly, the porous carbon filled with dextran gel was tested as a material for direct HPLC analysis of drugs in human serum.     

Purification and immobilization of Aspergillus niger β-xylosidase

β-Xylosidase from a commercial Aspergillus niger preparation was purified by differential ammonium sulfate precipitation and either gel permeation or cation exchange chromatography, giving 16-fold purification in 32% yield for the first technique or 27-fold purification in 19% yield for the second. The second method in addition almost completely removed interfering β-glucosidase activity. Enzymes prepared by this method was immobilized to 10 different carriers, but only when it was bound to alumina with TiCl₄ and to alkylamine porous silica with glutaraldehyde were substantial efficiencies and stabilities achieved. With alumina, the variation of activation procedure, amount of β-xylosidase offered, and activation solution composition yielded maximum activities of over 40 U/g with approximately 70% immobilization efficiency. Variation of binding pH and incubation time led to a maximum immobilized activity of 1.3 U/g with 78% immobilization efficiency on silica.     

Purification of human IgG by negative chromatography on ω-aminohexyl-agarose

The ω-aminohexyl diamine immobilized as ligand on CNBr- and bisoxirane-activated agarose gel was evaluated for the purification of human immunoglobulin G (IgG) from serum and plasma by negative affinity chromatography. The effects of matrix activation, buffer system, and feedstream on recovery and purity of IgG were studied. A one-step purification process using Hepes buffer at pH 6.8 allowed a similar recovery (69–76%) of the loaded IgG in the nonretained fractions for both matrices, but the purity was higher for epoxy-activated gel (electrophoretically homogeneous protein with a 6.5-fold purification). The IgG and human serum albumin (HSA) adsorption equilibrium studies showed that the adsorption isotherms of IgG and HSA obeyed the Langmuir–Freundlich and Langmuir models, respectively. The binding capacity of HSA was high (210.4 mg mL^?1 of gel) and a positive cooperativity was observed for IgG binding. These results indicate that immobilizing ω-aminohexyl using bisoxirane as coupling agent is a useful strategy for rapid purification of IgG from human serum and plasma.     

Regulation by Mg2+ and Ca2+ of mitochondrial membrane integrity: study of the effects of a cytosolic molecule and Ca2+ antagonists.

The coupling of aliphatic amines to agarose by the cyanogen bromide reaction yields isourea linkages which are positively charged at pH 7. The presence of these cationic sites in affinity gels causes significant non-specific adsorption of proteins. Serum albumin was found to bind to a number of derivatized gels which possessed these charged groups. The use of adipic dihydrazide as the leash moiety yielded affinity gels which were noncharged at pH 7. Serum albumin failed to adsorb to these gels. Beta-galactosidase from Escherichia coli was found to be sensitive to both ionic and hydrophobic groups in an affinity gel. A sample of active-site inhibited enzyme was found to bind to an affinity gel which contained both the cationic isourea and a phenyl structure in the leash. Thus it was concluded that the affinity purification of this enzyme has yet to be demonstrated. These studies dictate against the use of salt and pH gradients to desorb enzymes from affinity sorbents.     

The isolation and characterization of a colony stimulating factor from human lung.

Serum-free conditioned medium from human lung obtained at autopsy provides a rich source of colony stimulating factor which stimulates granulocytic and macrophagic colony growth in both mouse and human bone marrow. The appearance of the factor is enhanced by endotoxin and inhibited by either puromycin or actinomycin D. Human lung colony stimulating factor is stable at the pH range of 6.5-10 and temperature of 56 degrees C for 30 min. It is resistant to trypsin and neuraminidase but is sensitive to subtilisin, chymotrypsin and periodate. It shows heterogeneity on Sephadex gel filtration with two activity peaks having molecular weight of 200 000 and 40 000, respectively. Upon gel electrophoresis, human lung colony stimulating factor migrates in the alpha-globulin post-albumin region. Using the combination procedures of hydroxyapatite chromatography and preparative polyacrylamide gel electrophoresis a 600-fold purification was achieved with a final specific activity of 6-10(5) units per mg protein. The purified colony stimulating factor is very labile; however, the activity can be stabilized by the addition of gelatin or bovine serum albumin at the concentration of 0.1% and 0.2 mg/ml, respectively.     

Interaction of human platelet membrane glycoproteins with collagen and lectins

The binding of platelets to collagen is the first step in hemostasis. We attempted three approaches for elucidation of the chemical nature of receptors of human platelets for collagen. First, we examined the effect of platelet surface alteration by chymotrypsin treatment. On increasing the concentration of chymotrypsin, collagen-induced platelet aggregation and the release reaction decreased, and in parallel with this change, remarkable decrease of membrane glycoproteins IIb and V, as well as 400 kDa and 300 kDa membrane proteins, was observed. Secondly, effects of several lectins on the platelet-collagen interaction were examined. Lens culinaris agglutinin was found to specifically inhibit the platelet aggregation and release reaction induced by collagen. This inhibition appeared to be caused mainly by blocking of the collagen receptors on platelets by Lens culinaris agglutinin. Furthermore, Lens culinaris agglutinin was found to bind preferentially to glycoprotein IIb as identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of platelet membranes followed by staining with ¹²⁵I-Lens culinaris agglutinin. In addition, a polymerized preparation of Lens culinaris agglutinin induced platelet aggregation. Thirdly, the membrane component which could bind to collagen-Sepharose 4B was determined. Analysis by SDS-polyacrylamide gel electrophoresis combined with autoradiography or fluorography revealed that glycoprotein IIb was most enriched in the bound fraction to collagen. From these results, glycoprotein IIb is most likely a receptor for collagen on human platelet membranes.     

New matrices for the purification of pectinases by affinity chromatography

Polygalacturonic acid was used as a ligand in the affinity technique for pectinases purification from the filtrate of Aspergillus niger 71 culture. For this purpose four matrices were examined, namely, alkylamine controlled porous glass (CPG), alkylamine silica gel as well as keratin or polyamide coated silica gel. Good results of pectinase purification was obtained on silanized CPG or keratin coated silica gel supports.     

γ-Glutamyl transpeptidase of the ackee plant

The enzyme γ-glutamyl transpeptidase was purified from seeds of immature ackee fruit (Blighia sapida; Sapindaceae) by salt fractionation and gel filtration on Biogel P-10 and P-200. The procedure, which differs from an earlier one applied to kidney bean fruit, achieves 9.8% yield and 577-fold purification. The enzyme is also present in other parts of the fruit and in leaves. A MW of 12 500 was found by SDS-polyacrylamide gel electrophoresis, a value much lower that that reported for the enzyme from kidney bean fruit. Neutral or amino sugar accounts for 10% of the dry weight. In vitro, the enzyme catalysed synthesis of an unusual γ-glutamyl dipeptide which occurs in ackee seeds, using glutathione as glutamyl group donor. The enzyme mechanism was of the double displacement (ping-pong) type.