Interaction of pigeon-liver nicotinamide-adenine dinucleotide kinase with cibacron blue F3GA.

The interaction of pigeon liver NAD kinase with Cibacron Blue F3GA was investigated. By using steady-state rate measurements, spectrophotometric titration and chromatography of the enzyme on immobilized dye, it was shown that binding occurs at two nucleotide sites with different affinities, and also at a site distinct from the substrate-binding region.     

Specific interaction of spermine with cibacron blue F3GA

The polyamines, spermine, spermidine, and putrescine, have been shown to bind to Cibacron blue F3GA generating a difference spectrum with a maximum at 685 nm and a minimum at 585 nm, which is characteristic of ionic interactions between the dye and the polyamines. The difference spectral signal vanishes when the charges on the amino groups of the polyamines are neutralized. The magnitude of perturbation of the dye spectrum by the polyamines and, by inference, the capacity to bind to the dye, decrease in the order spermine > spermidine > putrescine. For spermine, the spectral signal of the dye-spermine complex is dependent on the charge state of an aminium group with a pK_a = 8.2.     

Interaction of vitamin D-binding protein with immobilized cibacron blue F3-GA.

An interaction of vitamin D-binding protein to immobilized Cibacron Blue F3-GA was studied under urea containing buffers. In these buffers, this protein was adsorbed to the immobilized dye and was eluted with salt gradients as in the same buffer without urea. The protein was also adsorbed to immobilized diethylaminoethyl but not to immobilized carboxymethyl. It is implicated that a combination of pseudo-ligand affinity and/or hydrogen bonding interaction plays a large role whereas ionic, hydrophobic and lipophilic interactions act little between the protein and the immobilized blue dye.     

ELISA-based quantification of cibacron blue F3GA used as ligand in affinity chromatography.

The development of an enzyme-linked immunosorbent assay (ELISA) for Cibacron Blue F3GA is reported. It quantifies a trace amount of blue dye used as an affinity chromatography ligand, rendering possible the measurement of traces of leached dye. Polyclonal antibodies were prepared after conjugation on the dye on KLH and injection into rabbits. The ELISA test was based on the competitive inhibition between a hemoglobin-dye complex adsorbed on the wells of a titration microplate and a free dye. The sensitivity of the assay was about 1000 times higher than a classical spectrophotometric assay and was modulated by some chemical substituents to the dye. The described ELISA assay was also successfully applied to the quantification of dye traces in the presence of human albumin.     

Predicting the binding properties of cibacron blue F3GA in affinity separation systems

The binding properties of cibacron blue F3GA (CB-F3GA) bound to a model NAD(P)H/FAD(H2)-dependent protein system, namely cytosolic quinone reductase (QR), was characterized by AMBER in an attempt to address the binding properties of immobilized CB-F3GA used in the separation of serum albumin. A favorable binding free energy of -4.52kcal/mol (KD=5.09 x 10(-4)kcal/mol) was determined for CB-F3GA binding by MM-PBSA method, which was found to be a ballpark estimate of empirical values reported in literature (DeltaG approximately -6kcal/mol). We propose that CB-F3GA primarily follows a class III binding motif in presence of FAD in the binding site of QR in solution, while a class II binding motif is observed in the crystal form. It was found that favorable van der Waals/hydrophobic interactions take place in the binding site making a major contribution to a favorably dominating enthalpy of binding (DeltaHtot=-25.87kcal/mol) as compared to a disfavorable binding entropy term (TDeltaStot=-21.35kcal/mol). Additional MM-PBSA experiments in the absence of FAD gave rise to a disfavorable binding free energy for CB in complex with QR, suggesting that FAD is an essential determinant of CB-F3GA binding. This is in contrast to an earlier observation of Denizli et al. on separation of human serum albumin (HSA) by immobilized CB-F3GA in the absence of FAD. Therefore, a class I binding model for CB-F3GA is proposed here to account for the efficient separation of HSA in affinity chromatography systems.     

Interaction of 3-hydroxybenzoate-6-hydroxylase with cibacron blue

Cibacron blue is a potent inhibitor of 3-HBA-6-hydroxylase at a concentration < 1 microM. Kinetic analyses revealed that at a concentration below 0.5 microM the dye behaves as an uncompetitive inhibitor with respect to 3-HBA and competes with NADH for the same site on the enzyme. The alteration of the near-UV CD spectrum and quenching of the emission fluorescence of the enzyme by cibacron blue indicates a significant alteration in the environment of aromatic amino acid residues due to a stacking interaction and subtle conformatiodnal changes in the enzyme. The concentration-dependent quenching of the intrinsic fluorescence of the enzyme by cibacron blue was employed to determine the binding parameters such as association constant (Ka) and stoichiometry (r) for the enzyme-dye complex.     

Interaction of 3-hydroxybenzoate-6-hydroxylase with cibacron blue

Cibacron blue is a potent inhibitor of 3-HBA-6-hydroxylase at a concentration < 1 μM. Kinetic analyses revealed that at a concentration below 0.5 μM the dye behaves as an uncompetitive inhibitor with respect to 3-HBA and competes with NADH for the same site on the enzyme. The alteration of the near-UV CD spectrum and quenching of the emission fluorescence of the enzyme by cibacron blue indicates a significant alteration in the environment of aromatic amino acid residues due to a stacking interaction and subtle conformatiodnal changes in the enzyme. The concentration-dependent quenching of the intrinsic fluorescence of the enzyme by cibacron blue was employed to determine the binding parameters such as association constant (K_a) and stoichiometry (r) for the enzyme-dye complex.     

Stimulation of bovine sperm motility and respiration by the triazine dye cibacron blue F3GA

Bovine sperm motility and respiration were stimulated by the triazine dye Cibacron Blue F3GA (CB), which may operate as a nucleotide mimic. CB stimulation of respiration was half-maximal at about 35 μM and respiration reached maximal levels about 1.5 minutes after CB addition. Respiratory stimulation was preceded by a transient increase in cytosolic cAMP. Sperm cAMP titers were elevated from 5 to 10 pmoles/10⁸ cells within 30 seconds of CB addition, but rapidly dropped to a stable level of about 7.5 pmoles/10⁸ cells. CB was a potent inhibitor of sperm membrane adenylyl cyclase and inhibited respiration in permeabilized cells. Taken together, the data indicated that CB stimulation was not manifested via the cytosol. In addition, a nonpermeant blue dextran preparation synthesized with CB also stimulated sperm respiration and motility. CB inhibited sperm membrane phosphodiesterase activity, suggesting that the transient pulse of cAMP resulted from CB interaction with this enzyme in the sperm membrane. © 1995 wiley-Liss, Inc.     

Interactions of contractile proteins with free and immobilized cibacron Blue F3GA.

Differential binding of contractile proteins from skeletal muscle to Cibacron Blue F3GA-Sepharose affinity columns provides the basis for a new purification technique. Myosin subfragments bind at low ionic strength and are eluted by high salt (e.g., 1.5 m NaCl). Myosin light chain 2 also binds at low ionic strength, whereas light chain 1 is only partially retarded and light chain 3 does not bind. Myosin's marginal solubility in the low-salt buffers required for binding renders it unsuitable for Blue Sepharose chromatography. Neither G-actin nor F-actin bind. Crude preparations of myosin subfragment-1 or light chains undergo significant purification upon Blue Sepharose chromatography. Nee free chromophore inhibits the ATPase activities of myosin and actomyosin at micromolar dye concentrations, whereas the binding of subfragment-1 to actin (in myofibrils) and the tension of glycerinated fibers are inhibited at millimolar dye concentrations. The dye binds at multiple sites on myosin, and inhibits its actomyosin ATPase both competitively and uncompetitively.     

Different profiles of induced cotton effects of human and bovine lactoferrin by cibacron blue F3GA binding.

1. The properties of complex formation of lactoferrin with Cibacron Blue F3GA dye have been studied by circular dichroic spectral analyses. The Cotton effects were induced by the interaction of lactoferrin with the dye and occurred in the wavelength range from 300 to 450 nm. 2. The patterns of changes in circular dichroic spectra of lactoferrin induced by the dye were different in bovine and human lactoferrin. 3. Iron ions bound to lactoferrin affected the profiles of induced Cotton effects in human lactoferrin but not in bovine lactoferrin. 4. While the dye bound co-operatively to human apo-lactoferrin, such co-operativeness was not observed in iron-saturated human lactoferrin.     

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.     

Investigation into the use of graft copolymer-based cibacron blue F3GA columns for the purification of proteins

The graft copolymers Nylon-co-hydroxyethylmethacrylate and poly(ethylene)-co-hydroxyethylmethacrylate coupled to Cibacron blue F3GA at wet volume levels similar to those obtained with Sepharose 4B. However, the graft copolymers removed protein from human serum to a far lesser extent than did Sepharose 4B. Further investigations involved the preparation of hydrolyzed poly(vinyl acetate) copolymers of nylon and polyethylene and of cellulose-co-hydroxyethylmethacrylate and study of the ability of the copolymers to remove human serum albumin and lactic dehydrogenase. Comparisons were made with Sepharose 4B-, Sephadex G15-, and G25-based Cibacron blue F3GA systems. The effectiveness of Sepharose 4B-Cibacron blue F3GA is thought to be due to the manner in which the dye is located within the pores of the gel.     

Characterization of fragments of human albumin purified by Cibacron blue F3GA affinity chromatography.

Controlled limited proteolysis of human plasma albumin (0.3 mM; 37 degrees C; 15 min; pH 3.7) with pepsin [pepsin/albumin, 1:1000 (w/w)] in the presence of octanoic acid (4.2 mM) yields at least 14 fragments in the range of 5000--56000 Da. By utilizing a combination of conventional and affinity-chromatographic procedures, two fragments with mol. wts. 25000 and 27000 were purified to more than 99% homogeneity. The larger fragment consists of a continuous polypeptide chain and has been shown to contain the primary bilirubin-binding site. The small fragment contains an internal cleavage site. On the basis of amino acid compositions, N-terminal sequences, C-terminal sequences, molecular weights and other internal markers the locations of these fragments within the known sequence of human albumin were determined to be residues 49--308 for the 27000 Da peptide and 309--585 for the 25000 Da peptide. Peptide 309--585 contains an internal cleavage site and appears to be missing residues 408--423. These non-overlapping fragments should be useful for investigations of individual ligand-binding sites and for the determination of antigenic sites.     

Human brain creatine kinase binding to immobilized cibacron blue F3GA: characterization and use in purification of the enzyme.

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) from adult human brain grey matter was purified by cibacron blue F3GA-Sepharose affinity chromatography. By gel electrophoresis of the purified enzyme under non-denaturing conditions a single protein band was observed. The dye-bound enzyme was eluted using its substrate, ATP. Reversibility of the binding of purified creatine kinase to blue Sepharose by ATP in a concentration-dependent manner indicated that the cibacron blue molecule which structurally mimics nucleotides occupied the substrate binding site of the enzyme. Also the marked dependence of enzyme binding to blue Sepharose on Mg2+ concentration suggested that Mg2+ ion is capable of combining with the dye moiety to form a site-specific binding complex that is similar to the physiological substrate of creatine kinase, namely Mg(2+)-ATP or Mg(2+)-ADP.     

Kinetic investigation of the interaction of cibacron blue F3G-A with phosphofructokinase from yeast.

Yeast phosphofructokinase is strongly inhibited by Cibacron Blue F3G-A. The inhibition is competitive in respect to the phosphate donor. Fructose 6-phosphate and ATP are able to abolish the dye-inhibition. Replacement of the strong inhibitor ATP by ITP as phosphate donor gives qualitatively analogous effects. The influence of Cibacron Blue F3G-A on the kinetic pattern of yeast phosphofructokinase can be described in terms of the kinetic model of Freyer et al. [8] if one assumes that the dye binds to the ATP-binding sites in a competitive manner.     

Studies on the kinetic sequence of in vitro ribosome assembly using cibacron blue F3GA as a general assembly inhibitor.

We have found that all E. coli ribosomal proteins strongly bind to an agarose affinity column derivatized with the dye Cibacron Blue F3GA. We have also shown that the capacity to bind the dye is lost when the proteins are organized within the structure of the ribosome or are members of pre-formed protein-RNA complexes. We conclude that the binding of ribosomal proteins to this dye involves specific protein-RNA recognition sites. These observations led us to discover that Cibacron Blue can be used to inhibit in vitro ribosome assembly at any stage of the assembly process. This has allowed us to determine a kinetic order of ribosome assembly.     

Characterization of lipase in reversed micelles formulated with cibacron blue F-3GA modified span 85

Sorbitan trioleate (Span 85) modified by Cibacron Blue F-3GA (CB) was prepared and used as an affinity surfactant to formulate a reversed micellar system for Candida rugosa lipase (CRL) solubilization. The system was characterized and evaluated by employing CRL-catalyzed hydrolysis of olive oil as a model reaction. The micellar hydrodynamic radius results reflected, to some extent, the redistribution of surfactant and water after enzyme addition, and the correlation between surfactant formulation, water content (W0), micellar size, and enzyme activity. An adequate modification density of CB was found to be important for the reversed micelles to retain enough hydration capacity and achieve high enzyme activity. Compared with the results in AOT-based reversed micelles, CRL in this micellar system exhibited a different activity behavior versus W0. The optimal pH and temperature of the encapsulated lipase remained unchanged, but the apparent activity was significantly higher than that of the native enzyme in bulk solution. Kinetic studies indicated that the encapsulated lipase in the reversed micelles of CB-formulated Span 85 followed the Michaelis-Menten equation. The Michaelis constant was found to decrease with increasing surfactant concentration, suggesting an increase of the enzyme affinity for the substrate. Stability of the lipase in the reversed micelles was negatively correlated to W0.