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.     

Substrate specificity of Gaucher spleen phosphoprotein phosphatase

The spleen in Gaucher's disease contains elevated levels of two distinct acid phosphatases. One of the isoenzymes, a tartrate-resistant type 5 acid phosphatase which we have designated SP_II acid phosphatase, possesses considerable phosphoprotein phosphatase activity. The enzyme dephosphorylates phosvitin and casein at specific rates (V) of 38.6 and 45.0 units/mg, respectively. The dephosphorylation of the oligophosphoproteins as well as various fragments of phosvitin, histories, and monophosphopeptides was studied kinetically. Positive cooperativity (Hill coefficient = 1.3–2.0) was observed for the dephosphorylation of phosvitin and casein as well as for the dephosphorylation of fragments of phosvitin which contained as few as two vicinal phosphoserine residues. In contrast, the hydrolysis of phosphomonoesters such as o-phosphorylserine or various monophosphopeptides exhibited typical Michaelis-Menten kinetics. Cooperativity appears to depend upon the substrate rather than the enzyme. The cooperativity of dephosphorylation was not affected by altering the secondary structure of phosvitin from a random to β conformation or by acetylation of the protein; however, acetylated phosvitin was dephosphorylated more rapidly (V = 50.8 units/mg) than native phosvitin indicating that the very basic phosphatase enzyme (pI = 8.5) prefers more acidic phosphoproteins as substrates rather than basic proteins such as histone (V= 0.0013 unit/mg). A monophosphohexa-peptide (V = 0.47 unit/mg) and monophosphoheptapeptide (V = 0.18 unit/mg) proved to be much poorer substrates than phosvitin, and monophosphoproteins such as glycogen phosphorylase, phosphorylase kinase, and glycogen synthase were not dephosphorylated by the enzyme. Although the phosphatase is active on monophosphopeptides and the presence of flanking amino acids considerably decreases the K_m of the enzyme for the phosphoserine residue (up to 100-fold), the enzyme appears to prefer peptide or protein substrates that contain two or more phosphoserine residues in close proximity. Finally, previous results showing the spleen phosphatase to be composed of 16,000- and 20,000-dalton subunits were apparently due to proteolysis during isolation since when 1.0 mm phenylmethylsulfonyl fluoride was included in the isolation media, the enzyme appeared as a single 35,000-dalton species when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Affinity extraction of BSA with reversed micellar system composed of unbound Cibacron Blue

Affinity Cibacron Blue 3GA (CB) dye in aqueous phase was directly transferred to the reversed micelles due to electrostatic interaction between anionic CB and cationic cetyltrimethylammonium bromide (CTAB). The bovine serum albumin (BSA) transfer to the reverse micelles increases significantly in a wide range of pH by the addition of a small amount of CB ( approximately 1.0-7.0% of the total surfactant concentration) to the aqueous phase. For pH < pI, the selectivity can be significantly improved with the presence of affinity CB because no BSA was extracted in the absence of CB. For backward extraction of BSA from the micellar phase with stripping aqueous solution, the addition of 2-propanol to the aqueous phase can recover almost all BSA (98.5%) extracted into the reverse micelles.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.     

Interaction between transcobalamin II and immobilized Cibacron Blue F3GA

Human serum transcobalamin II (TC II), a vitamin B₁₂ (Cbl) transport protein, complexes with Cibacron Blue F3GA, a reactive blue dye which can bind to proteins that require nucleotides as cofactors. Apo-TC II and holo-TC II both bind, but intrinsic factor (IF) and R-type binders of Cbl do not. Other mammalian species TC II also complex with the dye. Greater than 87% of the applied TC II-CN-[⁵⁷Co]Cbl remains bound to the dye even at pH 4.0. At pH values below this, the CN-[⁵⁷Co]Cbl dissociates off TC II which remains bound to the dye. High salt concentrations will break the TC II-dye complex. Ionic forces were considered not to be involved since complexing also occurred at pH 9.0, 2.5 pH units above the isoelectric point of TC II. Failure to dissociate the TC II-dye complex with 50% glycerol makes hydrophobic interactions unlikely. In addition to the potential uses of TC II-Cibacron Blue F3GA complexes in a total scheme for protein purification, the possibility that TC II is a nucleotide-requiring protein should be explored.     

Purification of Phytochrome by Affinity Chromatography on Agarose-Immobilized Cibacron Blue 3GA

The binding of phytochrome to Cibacron Blue 3GA was utilized to develop a new affinity purification procedure for phytochrome. Brushite-purified phytochrome from rye (Secale cereale c.v. Cougar) was bound to agarose-immobilized blue dye in 0.1 molar potassium phosphate (pH 7.8), contaminating proteins washed out with 0.5 molar KCl, and homogeneous phytochrome eluted with 10 millimolar flavin mononucleate. Ninety-five per cent of the phytochrome applied bound, and 60 to 65% was eluted, giving a 25 to 30% yield for the complete one-day procedure. Affinity-purified rye phytochrome was identical to conventionally purified phytochrome in its behavior on sodium dodecyl sulfate gels, in gel exclusion chromatography, in sedimentation in sucrose density gradients and in its spectral properties.     

Affinity adsorption of lysozyme on a macroligand prepared with Cibacron Blue 3GA attached to yeast cells

The objective of this study was the development of affinity adsorbent particles with the appropriate characteristics to be applied in protein purification using the affinity ultrafiltration method. To prepare affinity macroligands Cibacron Blue 3GA, as a ligand molecule, was immobilized by covalent bonding onto yeast cell walls, the support material or matrix. The maximum attachment of the ligand to the matrix was 212 μmol/g (ligand dry weight/yeast dry weight). Lysozyme was selected as the protein model for the adsorption studies. Its adsorption onto the matrix without ligand and matrix with attached ligand were investigated batch-wise. The adsorption equilibrium isotherms appeared to follow a typical Langmuir isotherm. The maximum adsorption capacity (q(m)) of the Cell-Cibacron macroligand for lysozyme was 110 mg/ml of wet macroligand. The adsorbent was also employed for the separation of lysozyme from hen egg white. High purity lysozyme was obtained.     

Affinity separation of human plasma gelsolin on Affi-Gel Blue

Human plasma gelsolin was specifically eluted with 1 mM adenosine 5'-triphosphate from an Affi-Gel Blue column. Since the ionic strength of sodium chloride required to elute the protein from the dye column was much higher than that of 1 mM adenosine 5'-triphosphate, the binding of plasma gelsolin with the dye-ligand appeared to be biospecific. Taking advantage of this affinity interaction, we have developed a revised purification method of human plasma gelsolin. The purification included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, Affi-Gel Blue chromatography, and Phenyl-Sepharose chromatography. The method allowed a reproducible purification of the protein to apparent homogeneity, producing a 331-fold purification with a yield of 6%.     

The effect of matrix on the binding of albumin to immobilized Cibacron Blue.

With Cibacron Blue as a ligand, the suitability of Sepharose, Sephacryl, cellulose and Ultrogel as column supports for the affinity chromatography of human serum albumin was examined. Sepharose and Sephacryl were the most effective matrices. Albumin was readily removed from such columns by using an electrophoretic desorption technique.     

Lipase purification by affinity precipitation with a thermo-responsive polymer immobilized Cibacron Blue F3GA ligand

A thermo-responsive polymer (P_NNB) was synthesized with lower critical solution temperature 27.5°C and over 95% recovery. The adsorption of porcine pancreatic lipase on Cibacron Blue F3GA-conjugated P_NNB (P_NNB-CB) closely followed the bi-Langmuir adsorption isotherm. The maximum adsorption capacity was found at pH 5.0, with a ligand density of 18.4 μmol/g polymers. The optimized eluent was a 0.01 M phosphate buffer solution at pH 8.0 containing 20% ethylene glycol. Six adsorptiondesorption recycles indicated excellent reusability of the affinity adsorbent. P_NNB-CB was applied to separate porcine pancreatic lipase from its crude material giving a lipase activity recovery of 81.6% with a 16-fold purification factor. Lipase could be purified to single-band purity, according to gel electrophoresis. The purification strategy is therefore feasible and efficient for purifying proteins of interest.     

Decolorization of the anthraquinone dye Cibacron Blue 3G-A with immobilized Coprinus cinereus in fluidized bed bioreactor

Coprinus cinereus, which was able to decolorize the anthraquinone dye Cibacron Blue 3G-A (CB) enzymatically, was used as a biocatalyst for the decolorization of synthetic solutions containing this reactive dye. Coprinus cinereus was immobilized in both calcium alginate and polyacrylamide gels, and was used for the decolorization of CB from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when Coprinus cinereus was entrapped in calcium alginate beads, and was of about 3.84 mg g(-1) h(-1) with a 50% conversion time (t1/2) of about 2.60 h. Moreover, immobilized fungal biomass in calcium alginate continuously decolorized CB even after 7 repeated experiments without significant loss of activity, while polyacrylamide-immobilized fungal biomass retained only 67% of its original activity. The effects of some physicochemical parameters such as temperature, pH and dye concentration on decolorization performance of isolated fungal strain were also investigated.     

Bilirubin removal from human plasma by Cibacron Blue F3GA using immobilized microporous affinity membranous capillary method

A novel affinity sorbent system for direct bilirubin removal from human plasma was developed. These new adsorbents comprise Cibacron Blue F3GA as the specific ligand, and microporous membranous poly(tetrafluoroethylene) capillary (modified by coating with a hydrophilic layer of poly(vinyl alcohol) after activation) as the carrier matrix. The affinity adsorbents carrying 126.5 micromol Cibacron Blue F3GA/g polymer was then used to remove bilirubin in a flow-injection system. Non-specific adsorption on the poly(vinyl alcohol) coated capillary remains low, and higher affinity adsorption capacity, of up to 76.2 mg/g polymer was obtained after dye immobilization. The bilirubin adsorption capacity of the affinity capillary decreased with increase in the recirculation rate of plasma. The adsorption capacity increased with increase the temperature while decreased with increase the ionic strength. The maximum adsorption was only observed in neutral solution (pH 6-7). The adsorption isotherm fitted the Langmuir model well. These new adsorbents have higher velocity of mass transfer, better adsorption capacity, less fouling, longer service life and good reusability. The results of blood tests suggested the dye affinity capillary has good blood compatibility.     

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.     

Affinity chromatography of mouse and rat alpha-fetoproteins on immobilized diethylstilbestrol]

Alpha-fetoproteins (AFP) from amniotic fluid of mouse and rat demonstrate high affinity and specificity during their binding with immobilized diethylstilbestrol, which allows to isolate these two proteins by one step using the method of affinity chromatography on Sepharose with immobilized diethylstilbestrol. Meanwhile the yield of mouse AFP was 42%, and rat AFP--75%. The preliminary incubation of the amniotic fluid of rat and mouse with free estradiol results in abrupt fall of AFP outcome, which may testify to the binding of estradiol and diethylstilbestrol by the same receptor sites on AFP molecule.     

Chromatography of plasma proteins on immobilized Cibacron Blue F3-GA. Mechanism of the molecular interaction.

Fractionation of plasma proteins on immobilized Cibacron Blue F3-GA (Affi-gel Blue) under different conditions of pH, ionic strength and temperature was studied. At acidic pH the unbound proteins were eluted in order of increasing pI (the Affi-gel Blue behaving as ion-exchanger); at basic pH and at low ionic strength they were eluted in order of decreasing molecular weight (separation by diffusion-exclusion). For the proteins that were either retarded in comparison with substances of similar molecular characteristics, or that were bound to the resin, pseudo-ligand affinity or hydrophobic interactions were also implicated.     

Affinity differences between some commercially available immobilized Con A with rat alpha-fetoprotein

Rat alpha-fetoprotein contains three Con A-affinity molecular variants evidenced by affino-immuno-electrophoresis, (a) Con A-non reactive (52 %), (b) Con A-weakly reactive (31 %), (c) Con A-reactive (17 %). Affinity chromatography on several commercially available Con A-linked agarose displays different alpha-fetoprotein affinity patterns. The Con A-weakly reactive variant can be either bound and eluted with the Con A-reactive fraction or unbound and eluted with the Con A-non reactive one. These differences of chromatographic behaviour should be taken into account in structural, biochemical and biological studies dealing with the Con A-affinity molecular variants of alpha-fetoprotein or of other glycoproteins.