On-line separation of solute mixtures using reciprocating size exclusion chromatography

Solutes of different size in a mixture solution were separated on-line, using a semi-continuous reciprocating size exclusion chromatography. The band of fast-moving large molecules was isolated during the first half cycle, while the band of slow-moving small molecules was isolated during the second half cycle. After 7 cycles of frontal mode operation, 89% of the Blue Dextran in the feed was isolated as a pure solution. Vitamin B₁₂ of constant concentration was also isolated as a pure solution.     

Colorimetric device for measurement of transvascular fluid flux in blood-perfused organs

The aim of this study was to develop a device capable of measuring transvascular fluid flux in blood-perfused organs. For any given blood flow through the organ (QT), transvascular flux (QF) can be considered as the fraction of QT exchange. Presumably, QF would change the background concentration of an impermeable tracer residing in the perfusate. Thus QF could be calculated from the relative changes in tracer concentration for any given QT. We have used Blue Dextran (1 g/l of blood) as the reference tracer. Because the minimum molecular weight of Blue Dextran is 2 X 10(6), we anticipated it to behave as an impermeable tracer in most organs. QF was simulated with continuous infusions of plasma, normal saline solution, and a 50% mixture of both. Changes in Blue Dextran concentration were continuously followed colorimetrically by changes in transmission of specific light at a wavelength of 632 nm. Because 632-nm light is affected by hematocrit and O2 saturation changes, two additional wavelengths were used: 815-nm, which is not affected by saturation or Blue Dextran concentration changes, was used to account for changes in hematocrit, and 887-nm specific light, which is not affected by Blue Dextran, served to correct for saturation changes. Red cells could not be used as the reference tracer because of the possibility of hematocrit changes independent of fluid flux (Fahraeus effect). The device so constructed proved capable of measuring rates of fluid infusion in the order of 0.1% of QT with a variability of 10% around the mean.     

Different interactions of human and bovine serum albumin with Cibacron Blue and Blue Dextran

The interaction of human and bovine serum albumin with Cibacron Blue and Blue Dextran in aqueous solution was studied by means of difference spectroscopy. Both human and bovine albumin interact strongly with underivatized Cibacron Blue in three independent binding sites (K = 105). On the contrary, Blue Dextran interacts strongly only with human albumin, but does not bind appreciably to bovine albumin. These results suggest that the binding sites are exposed and easily accessible in human albumin, while in bovine albumin they are sterically hindered and therefore not accessible to the bulky Blue Dextran.     

On-line recovery of large molecules and concentration of small molecules using reciprocating size exclusion chromatography with temperature swing

The reciprocating size exclusion chromatography (RSEC) was operated with swing between two temperatures in a synchronous way with flow direction to recover a large solute on-line from the mixture, in addition to the small solute concentration. The concentration of small solutes in RSEC with a temperature swing was made possible by taking advantage of the temperature-dependent swelling properties of the porous gel. After 7 cycles of frontal mode operation, 76% of Blue Dextran in the feed was recovered and nickel nitrate in the feed reservoir was concentrated by 13%.     

Affinity electrophoresis of proteins interacting with Blue dextran.

Interaction of several enzymes (pyruvate kinase, myokinase, creatine kinase, aldolase, malate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase) and other proteins (bovine serum albumin and ovalbumin) with Blue Dextran was studied by means of affinity electrophoresis in polyacrylamide gels. A decrease of electrophoretic mobility of enzymes in affinity gels was dependent on Blue Dextran concentration and in some cases, dissociation constants of the protein-immobilized dye complexes could be calculated. Affinity electrophoresis in the presence of Blue Dextran reveals in some cases additional bands of isoenzymes, as compared with the control gels (without Blue Dextran).     

Interaction of cibacron blue 3G-A and related dyes with nucleotide-requiring enzymes

Cibacron Blue 3G-A (I), the chromophore in Blue Dextran, its structural isomer Cibacron Brilliant Blue BR-P (II), and two other structural analogs (III, IV) were used to probe the nucleotide binding sites of selected kinases and dehydrogenases. Inhibition studies indicate that the portion of the dye molecule necessary for effective inhibition of nucleotide binding is a structure similar to 1-amino-4(4′-aminophenylamino)-anthraquinone-2,3′-disulfonic acid (ASSO; III). The strong inhibition exhibited by these dyes is likely to be due to interaction with specific nucleotide binding sites, irrespective of the presence of a “dinucleotide fold” in the proteins' supersecondary structure.     

Purification of dihydropterin reductase using immobilized Cibacron Blue.

Chromatography on columns of immobilized Cibacron Blue (Blue Dextran--agarose) can be used as a major step in the purification of quinonoid dihydropterin reductase. The reductase has been isolated from fractions of beef kidney by selective binding to the immobilized Cibacron in the presence of tetrahydropterin. The binding of the reductase to Blue Dextran and its specific elution from columns of Blue Dextran--agarose indicate that the reductase possesses the dinucleotide (NAD+) binding domain. The results of kinetic experiments give validity to both our affinity chromatography of the reductase and to an ordered mechanism for the formation of tetrahydropterin. Chromatography on Blue Dextran--agarose has been used to show that folate or amethopterin can compete with Cibacron Blue for the dinucleotide domain of the reductase. The p-aminobenzoyl-glutamate moiety of the folates competes with Cibacron Blue for the NADH site of the reductase. A stable binary complex of dihydropterin reductase with NADH has been detected by gel electrophoresis.     

Properties of carbohydrate-metabolizing enzymes immobilized in sol-gel beads: stabilization of invertase and β;-glucosidase by Blue Dextran**

When immobilized in sol-gels, invertase (;-fructofuranosidase) from Candida utilis and ;-glucosidase from Pyrococcus furiosus had activity recovery values of 30 and 28%, respectively. However, if Blue Dextran (0.04%) was included in the immobilization-reaction mixture, the respective recovery values increased to 63 and 52%. Glucose dehydrogenase from Thermoplasma acidophilum immobilized by the same method lost most of its activity and Blue Dextran had no effect on the recovery of activity during the immobilization procedure. The immobilized enzymes required treatment with glutaraldehyde in order to maintain their activity within the sol-gel matrix during continuous reaction with their respective substrates.     

Precipitation of soluble affinity complexes by a second affinity interaction: a model study

Lactate dehydrogenase was purified by affinity precipitation. The enzyme bound to Blue Dextran (the Cibacron blue residues) and was precipitated by addition of concanavalin A. The lectin functions as a crosslinking agent, building up large flocs of dextran that subsequently precipitate, thus co-precipitating the affinity-bound lactate dehydrogenase.     

A method using fibrin-fixed Blue Dextran for determining the plasmin and plasmin inhibitor activities in human plasma.

The fibrinolytic activity of plasmin was determined by incubating with fibrin-fixed Blue Dextran as a substrate, the Blue Dextran released being proportional to the plasmin activity. The applicability of this method for rapid and accurate evaluation of fibrinolytic activity was demonstrated by dose-response curves with purified plasmin, plasmin generated by urokinase in human plasma and euglobulin. The method can also be used to determined plasmin inhibitors in plasma.     

Affinity chromatography of Ankistrodesmus braunii nitrate reductase using blue dextran-sepharose (author's transl)

Blue Dextran has been coupled covalently to Sepharose-4B to purify the enzymatic complex NAD(P)H-nitrate reductase (EC 1.6.6.2) from the green alga Ankistrodesmus braunii by affinity chromatography. The optimum conditions for the accomplishment of the chromatographic process have been determined. The adsorption of nitrate reductase on Blue Dextran Sepharose is optimum when a phosphate buffer of low ionic strength and pH 6.5-7.0 is used. Once the enzyme has been bound to Blue Dextran Sepharose, it can be specifically eluted by addition of NADH and FAD to the washing buffer. However, none of the nucleotides added separately is able to promote the elution of the enzyme from the column. The elution can be also achieved, but not specifically, by increasing the ionic strength of the buffer with KCl. These results have made possible a procedure for the purification of A. braunii nitrate reductase which led to electrophoretic homogeneity, with an overall yield of 70% and a specific activity of 49 units/mg of protein.     

The Hydrolysis of Dextran by Gram Negative Non-sporing Anaerobic Bacilli

The ability of Gram negative anaerobic bacilli to hydrolyse dextran was determined in liquid and solid media containing Blue Dextran 2000. Released blue chromophore in the liquid medium was detected spectrophotometrically. Results obtained with 334 strains of Bacteroidaceae grown on the solid medium indicated that most strains did not hydrolyse the substrate. Hydrolysis of Blue Dextran 2000 occurred with certain strains of Bacteroides thetaiotaomicron , B. melaninogenicus ss. melaninogenicus, B. oralis, B. ovatus and B. ochraceus.     

Conversion of quinate to 3-dehydroshikimate by Ca-alginate-immobilized membrane of Gluconobacter oxydans IFO 3244 and subsequent asymmetric reduction of 3-dehydroshikimate to shikimate by immobilized cytoplasmic NADP-shikimate dehydrogenase

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.     

The specific interaction of Cibacron and related dyes with cyclic nucleotide phosphodiesterase and lactate dehydrogenase

1. Reactive Blue 2 (Cibacron Blue 3G-A) is a competitive inhibitor of bovine heart cyclic nucleotide phosphodiesterase (K(i) 0.3mum). The K(i) increases with increasing temperature, suggesting that hydrophobic interactions are not largely responsible for the binding of the dye. Another 25 sulphonated aromatic dyes are also competitive inhibitors of the cyclic nucleotide phosphodiesterase (K(i) values in the range of 0.06-13.6mum). 2. These dyes (covalently linked to Dextran 40) inhibit bovine heart cyclic nucleotide phosphodiesterase. Reactive Blue 2 (covalently linked to Dextran 40) is a competitive inhibitor of the phosphodiesterase (K(i) 0.4mum). 3. Bovine heart cyclic nucleotide phosphodiesterase is retained on a column of Reactive Blue 2-Sephacryl S-200 and can be eluted from the column by 3':5'-cyclic AMP. 4. A variety of the dyes (either free or covalently linked to Dextran 40) are competitive inhibitors of rabbit muscle lactate dehydrogenase. 5. The effectiveness of a wide range of structurally dissimilar dyes as competitive inhibitors of lactate dehydrogenase and cyclic nucleotide phosphodiesterase compromises proposals for the use of Reactive Blue 2 as a specific probe for the dinucleotide-binding structural domain present in many dehydrogenases and kinases. Detailed information of the various dyes used has been deposited as Supplementary Publication SUP 50089 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Partial characterization of the in vitro activation of inactive pyruvate, pi dikinase from darkened maize leaves

In vitro activation of dark-inactivated pyruvate, orthophosphate dikinase extracted from maize (Zea mays L. cv. Golden Cross Bantam T51) leaves was examined. The inactive form of the enzyme and orthophosphate behaved kinetically as substrates for the reaction, which was catalyzed by an activating factor. This factor was bound by Blue Dextran Sepharose 4B and could be eluted by KCl at a concentration of 0.5m. The molecular weight of the maize leaf activating factor was about 88,000. Cibacron Blue 3G-A, a reactive moiety of Blue Dextran, inhibited the factor competitively with respect to the concentration of the inactive dikinase with a K(i) of 4.6 micromolar. Adenosine diphosphate and pyrophosphate were also found to be competitive inhibitors of activation, with respect to the inactive dikinase, giving K(i) values of 90 and 140 micromolar, respectively. Adenosine, other nucleotide diphosphates, and dinucleotides gave little or no inhibition of activation. These results suggest the association of a nucleotide, presumably nucleotide diphosphate, with the inactive form of pyruvate, orthophosphate dikinase.     

Conversion of Quinate to 3-Dehydroshikimate by Ca-Alginate-Immobilized Membrane of Gluconobacter oxydans IFO 3244 and Subsequent Asymmetric Reduction of 3-Dehydroshikimate to Shikimate by Immobilized Cytoplasmic NADP-Shikimate Dehydrogenase

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.     

The origin and consequences of concentration dependence in gel chromatography

Concentration dependence of elution volume was determined for Blue Dextran 2000, Dextran 500, Dextran sulphate 500 and bovine serum albumin on columns of Sephadex G-100 equilibrated with sodium phosphate buffer, I 0.1, pH6.8. From the results for Dextran 500, it was shown that a linear relation exists between elution volume and the corresponding osmotic pressure calculated for the same concentration and incorporating the term containing the second virial coefficient. This relationship was used to predict the concentration dependence of elution volume for bovine serum albumin and myoglobin, proteins that partially penetrate Sephadex G-100. Possible consequences of osmotic effects are considered in relation to various types of column experiments, including differential chromatography.     

Stabilization of basic fibroblast growth factor with dextran sulfate.

Dextran sulfate protected bFGF from heat and acid inactivation and from proteolytic degradation. The protective effect was stronger than that of heparin which is known as a stabilizer of bFGF. Dextran sulfate and bFGF formed a high molecular weight complex via ionic interaction when mixed together in aqueous solution. The complex was dissociated when the ionic strength was increased and the protective effect was completely abolished. Successive digestion of bFGF with Staphylococcus aureus V8 protease and pepsin followed by affinity chromatography on an immobilized dextran sulfate column and reversed-phase high performance liquid chromatography yielded three positively charged fragment peptides, Tyr24-Phe30, Tyr106-Trp114 and Tyr124-Leu138. These results suggest that dextran sulfate stabilizes bFGF by binding close to the putative heparin binding sites of the bFGF molecule.     

Acetyl-coenzyme A and coenzyme A analogues. Their effects on rat brain choline acetyltransferase.

Choline acetyltransferase has the same affinity for acetyl-CoA, propionyl-CoA and butyryl-CoA (Km=1.4 micron). Choline acetyltransferase may use the two latter compounds as substrate, but the longer the acyl chain the lower will be Vmax. CoA is an inhibitor (Ki=1.8 micron). The position of the 3'-phosphate is of primary importance. Desphospho-CoA is a weak inhibitor (Ki=500 micron). 5'-AMP is already an inhibitor (Ki=2500 micron). Phosphopantetheine is not an inhibitor. Dextran Blue is a potent inhibitor (Ki=0.05 micron). Choline acetyltransferase binds to hydrophobic affinity columns. Because of its affinity for nucleotides, affinity for Dextran Blue and hydrophobicity, it is proposed that it contains the 'nucleotide fold', which is a common structural domain present in several enzymes binding nucleotides.     

Rapid purification and properties of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae.

A method for the purification of yeast K+-activated aldehyde dehydrogenase is presented which can be completed in substantially less time than other published procedures. The enzyme has a different N-terminal amino acid from preparations previously reported, and other small differences in amino acid content. These differences may be the result of differential proteolytic digestion rather than a different protein in vivo. A purification step involves the biospecific adsorption on affinity columns containing immobilized nucleotides in the absence of the substrate aldehyde. Direct binding studies with the coenzyme in the absence of aldehyde reveal 4 NAD sites per tetrameric molecule, each with a dissociation constant of 120 micron. These results conflict with properties of preparations previously reported and may conflict with kinetic models that have aldehyde as the leading substrate. Binding to Blue Dextran affinity columns suggests the presence of a dinucleotide fold in common with other dehydrogenases and kinases.