Blue Sepharose CL-6B Affinity Chromatography Research Articles

Structural and Catalytic Properties of the D-3-Hydroxybutyrate Dehydrogenase from Pseudomonas aeruginosa

To put forward BDH from Pseudomonas aeruginosa's enzymatic properties, we report a two-step purification of BDH and its gene sequencing allowing the investigation of its structural properties. Purification of BDH was achieved, using ammonium sulfate fractionation and Blue Sepharose CL-6B affinity chromatography. SDS-PAGE analysis reveals a MM of 29 kDa, whereas the native enzyme showed a MM of 120 kDa suggesting a homotetrameric structure. BDH encoding gene sequence shows a nucleotide open reading frame sequence of 771 bp encoding a 265 amino acid residues polypeptide chain. The modeling analysis of the three dimensional structure fits with the importance of amino acids in the catalysis reaction especially a strictly conserved tetrad. Amino-acid residues in interaction with the coenzyme NAD(+) were also identified.

Purification of alpha-sarcin and an antifungal protein from Aspergillus giganteus by blue sepharose CL-6B affinity chromatography.

A simple method for preparation of alpha-sarcin and an antifungal protein (AFP) from the mold Aspergillus giganteus MDH 18894 has been developed. alpha-Sarcin and AFP were purified simultaneously by carboxymethylcellulose 52 cation-exchange chromatography and Blue Sepharose CL-6B affinity chromatography. By this method, 4.2 mg of alpha-sarcin and 6.8 mg of AFP were obtained from 2 liters of medium. Compared with other purification methods such as gel-filtration chromatography, this procedure was simple and specific. The purified alpha-sarcin and AFP were homogeneous characterized on SDS-polyacrylamide gel. The enzymatic activity of several ribosome-inactivating proteins such as alpha-sarcin, trichosanthin, and cinnamomin was significantly inhibited by the dye Cibacron blue F3GA. In 50 microliter of reaction mixture, 10 microM of the dye could inhibit 50% activity of cinnamomin (7 x 10(-9) M), whereas 50% inhibition of the enzymatic activity of trichosanthin (7 x 10(-9) M) and alpha-sarcin (1 x 10(-7) M) required 100 and 50 microM of the dye, respectively.

Purification and properties of NAD(P)H: (quinone-acceptor) oxidoreductase of sugarbeet cells.

NAD(P)H:(quinone-acceptor) oxidoreductase [NAD(P)H-QR], a plant cytosolic protein, was purified from cultured sugarbeet cells by a combination of ammonium sulfate fractionation, FPLC Superdex 200 gel filtration, Q-Sepharose anion-exchange chromatography, and a final Blue Sepharose CL-6B affinity chromatography with an NADPH gradient. The subunit molecular mass is 24 kDa and the active protein (94 kDa) is a tetramer. The isoelectric point is 4.9. The enzyme was characterized by ping-pong kinetics and extremely elevated catalytic capacity. It prefers NADPH over NADH as electron donor (kcat/Km ratios of 1.7 x 10(8) M-1 S-1 and 8.3 x 10(7) M-1 S-1 for NADPH and NADH, respectively, with benzoquinone as electron acceptor). The acridone derivative 7-iodo-acridone-4-carboxylic acid is an efficient inhibitor (I0.5 = 5 x 10(-5) M), dicumarol is weakly inhibitory. The best acceptor substances are hydrophilic, short-chain quinones such as ubiquinone-0 (Q-0), benzoquinone and menadione, followed by duroquinone and ferricyanide, whereas hydrophobic quinones, cytochrome c and oxygen are reduced at negligible rates at best. Quinone acceptors are reduced by a two-electron reaction with no apparent release of free semiquinonic intermediates. This and the above properties suggest some relationship of NAD(P)H-QR to DT-diaphorase, an animal flavoprotein which, however, has distinct structural properties and is strongly inhibited by dicumarol. It is proposed that NAD(P)H-QR by scavenging unreduced quinones and making them prone to conjugation may act in plant tissues as a functional equivalent of DT-diaphorase.

Characterization of NADH-dependent Fe3+-chelate reductases of maize roots

Iron-deficient maize seedlings exhibit a starvation syndrome characterized by an increase in different parameters such as root fresh weight (+ 30%), protein (+ 25%) and plasma membrane-associated NADH Fe3+ −EDTA reductase (NFR; +45%). NFR activity was found associated with 9 000g (20 min) and 110 000 g (1 h) sediments, purified plasma membrane and 110000 g supernatants. No differences were observed between the properties of reductases from Fe-starved versus Fe-sufficient roots. The characterization of NFR was undertaken. Low Mr forms (46 and 28 kDa, as detected by size-exclusion chromatography) were present in all fractions whereas 210 and 110 kDa forms were unique in membranes and 110 000 g supernatants, respectively. The 210 kDa form was solubilized from microsomes and characterized. The enzyme is cetone-resistant and appears to be comprised largely if not totally of the low Mr forms (46 and 28 kDa, corresponding to 30 and 32 kDa bands, respectively, in SDS-PAGE). The 210 kDa form tended to break down to subunits following dilution, and the effect could be prevented by addition of 10% (v/v) glycerol. A three-step purification procedure for microsomal NFR was devised, consisting of acetone fractionation of lysophosphatidycholine solubilized microsomes, Blue Sepharose CL-6B affinity chromatography and a final size exclusion chromatography in the absence of detergent, resulting in a 700-fold purification of the 28 kDa protein. The best electron acceptor for the purified 28 kDa form was ferricyanide (400μmol min−1mg−1 protein) followed by Fe3+−chelates (up to 200μol min−1 mg−1 protein) and other compounds to a lesser extent (cyt c, DCPIP).The 46 kDa form, on the other hand, had high ferricyanide reductase activity (about 300μmol min−1mg−1 protein) and relatively low Fe3+−chelate reductase activity. The properties of NFR (high M, active forms, donor and acceptor specificity, purification behaviour, large hydrophilic domains, size of subunits) suggest a relationship with the NADH-cyt b5 reductase family of FAD-containing proteins. None of the latter flavoproteins is a transmembrane enzyme.

Molecular and biological properties of an abrin A chain immunotoxin designed for therapy of human small cell lung cancer.

An immunotoxin (IT) comprising abrin A chain attached to the mouse monoclonal antibody SWA11, recognising a cell surface antigen highly associated with human small cell lung cancer (SCLC), was synthesised using a hindered disulphide crosslinker, N-succinimidyl 3-(2-pyridyldithio) butyrate (SPDB), and purified by Blue Sepharose CL-6B affinity chromatography. The IT preparation contained monomeric conjugate, composed of one abrin A chain molecule linked to one SWA11 molecule, and was free from unconjugated A chain or antibody. The IT fully retained the cell-binding capacity of the antibody component and the ribosome-inactivating activity of the A chain. In cytotoxicity assays using the SW2 SCLC cell line in tissue culture, SWA11-SPDB-abrin A chain inhibited the incorporation of 3H-leucine by 50% at a concentration of 10 pM and by 99% at a concentration of 1 nM. The anti-tumour efficacy of the IT was tested in nude mice bearing established s.c. solid SW2 tumour xenografts. A single i.v. injection of SWA11-SPDB-abrin A chain at a non-toxic dose induced a significant 7 to 10 day growth delay that could not be matched by administration of equivalent doses of either unconjugated SWA11 or abrin A chain alone. The results of this study indicate that the antigen recognised by SWA11 is an effective target for therapy of SCLC with A chain ITs in vivo.

Purification and properties of .ALPHA.1- and .ALPHA.2-macroglobulins in the dog.

1) The ultrafiltration of serum collected from dogs by a filtration film with a pore size of 300, 000, Blue Sepharose CL-6B affinity chromatography and Ultrogel AcA 22gel filtration chromatography eluted a third protein fraction in addition to α1-macroglobulin (α1-M) and α2-macrogrobulin (α2-M). The subsequent use of preparative agarose gel isoelectric focusing with a cover gel facilitated the extraction of pure α1-M and α2-M protein fractions.2) Analytical agarose gel isoelectric focusing and Ferguson plot by agarose gel electrophoresis revealed the following: (1) dog α1-M had an isoelectric point (pI) of 5.4 and a molecular weight of 724, 000; and (2) dog α2-M had a pI of 5.7 and a molecular weight of 758, 000.

Purification of immunotoxins containing the ribosome-inactivating proteins gelonin and momordin using high performance liquid immunoaffinity chromatography compared with Blue Sepharose CL-6B affinity chromatography

Comparable amounts of the ribosome-inactivating proteins (RIPs) ricin A chain, gelonin and momordin were allowed to bind to Blue Sepharose CL-6B (immobilised Cibacron Blue F3GA) in phosphate buffer, pH 7.5, and were then eluted quantitatively with buffer containing 0.5 M NaCl. Differences in the elution profiles indicated that the RIPs possessed different affinities for the Cibacron Blue F3GA dye. • Conjugation of the RIPs to the monoclonal antibody Fib75 resulted in decreased affinity for Blue Sepharose. Under conditions allowing the complete separation of the Fib75-ricin A chain immunotoxin from unconjugated Fib75, the Fib75 immunotoxins made with gelonin and momordin failed to bind completely to the Blue Sepharose column. The Fib75-gelonin and Fib75-momordin fractions that eluted from the column with 0.5 M NaC1 were free of unconjugated Fib75 but were enriched in multiply substituted conjugate molecules. • A high performance liquidd immunoaffinity chromatography procedure based on the selective binding of conjugated RIP to immobilised affinity-purified anti-RIP antibody permitted the complete separation of the gelonin and momordin immunotoxins from unconjugated Fib75 without altering the composition, molecular integrity or cytotoxic activity of the immunotoxins.

Selective purification of sex‐influenced esterase from rat serum by immunoaffinity chromatographies

Sex-influenced esterase (ES-SI) is a female-specific serum esterase of rats. There are two alleles at the Es-Si locus, Es-Sia coding for ES-SI and Es-Sib, a silent allele. ES-SI was purified to electrophoretic homogeneity from the serum of adult female SI3 rats by a four-step purification. First, albumin was removed from the serum by Blue-Sepharose CL-6B affinity chromatography. Sera of rats carrying Es-Sia and Es-Sib were utilized to make two novel immunoaffinity columns. Rat anti-(ES-SI) alloantibody was obtained after the immunization of rats carrying Es-Sib with rat ES-SI-positive serum. ES-SI bound to the gel in this antibody-conjugated affinity chromatography. Simultaneously, rabbit anti-(rat serum protein lacking ES-SI) antibody was raised against ES-SI-negative rat serum and used to exclude other serum proteins. ES-SI was recovered in the flow-through fractions in this antibody-conjugated immunoaffinity chromatography, whereas the other serum proteins bound to the gel. ES-SI was selectively purified about 240-fold by combining these immunoaffinity chromatographies. This combination is a very powerful technique for purification of the protein. Finally, ES-SI was purified by preparative polyacrylamide gel electrophoresis after which it appeared as a single protein band (using silver staining) on SDS/polyacrylamide gel electrophoresis with a molecular mass of 72 kDa. In the ordinary estimation of enzyme purification, the purification of ES-SI was 30-fold at most, because the specific activity of the enzyme was calculated from the bulky esterase activity. However, ES-SI was selectively purified 2280-fold comparing its recovery to that of total protein. ES-SI exhibited a pI of 4.6 and optimum pH of 6.7. The activity of ES-SI was completely inhibited by diisopropyl fluorophosphate at 10 nM but not at all by eserine.

Purification of rat retinol-binding protein using Blue Sepharose CL-6B.

Studies were conducted to isolate rat plasma retinol-binding protein, the specific transport protein for vitamin A in the rat. Retinol-binding protein was isolated from rat plasma by a new simple procedure using Blue Sepharose CL-6B, and highly purified retinol-binding protein was obtained. This procedure included DEAE-cellulose chromatography at pH 6.0, Sephadex G-75 gel filtration in the presence of 3.0 M urea, Blue Sepharose CL-6B affinity chromatography at pH 7.0 and finally Sephadex G-100 gel filtration at pH 7.4. The third step completely accomplished the dissociation and separation of retinol-binding protein from its complex with prealbumin and plasma albumin. This procedure is a significant improvement over previously published procedures, in which sample recycling and preparative polyacrylamide gel electrophoresis are necessary. The molecular weight, electrophoretic behaviour, ultraviolet and fluorescence spectra of the retinol-binding protein were similar to those appearing in other reports.

The identification and characterization of two separate carboxylesterases in guinea-pig serum.

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.

Allosteric regulation of serine hydroxymethyltransferase from mung bean ( [formula omitted

Serine hydroxymethyltransferase, the first enzyme in the pathway for the interconversion of one carbon compounds was purified from mung bean seedlings by ammonium sulfate fractionation, DEAE-Sephadex, Blue Sepharose CL-6B affinity chromatography and gel filteration on Sephacryl S-200. The specific activity of the enzyme, 0.73 (u mol HCHO formed/min/mg protein) was 10 4 times larger than the highest value reported hitherto. Saturation of tetrahydrofolate was sigmoid, whereas with serine was hyperbolic, with n H values of 1.9 and 1.0 respectively. Reduced nicotinamide adenine dinucleotide, lysine and methionine decreased, whereas nicotinamide adenine dinucleotide, adenosine 5′-monophosphate and adenosine 5′-triphosphate increased the sigmoidicity. These results suggest that serine hydroxymethyltransferase from mung bean is a regulatory enzyme.