Dye-promoted precipitation of serum proteins. Mechanism and application.

Immobilized dyes have been used primarily for purification of nucleotide dependent enzymes and proteins from plasma and other sources. Due to their low costs, high protein binding capacity and resistance to degradation dyes bear the potential as ligand for affinity separation of proteins on a large scale. In this paper dyes have been used for precipitation of proteins. Using albumin, prealbumin, alpha 1-acid glycoprotein and immunoglobulin G as model proteins we could demonstrate that dye-promoted precipitation depends on several factors which include the structure of the dye, the pH of the solution, the dye/protein molar ratio and the intrinsic properties of the proteins. It revealed that most of the dyes tested were endowed with the precipitating potential. The efficacy of precipitation was found to increase with the complexity of the dye structure. However, the amount of a dye required for total precipitation was found to be different for a given protein. Electrostatic as well as hydrophobic forces are involved in the mechanism of precipitation. It was demonstrated that by optimizing the conditions, mixtures of proteins can be resolved by dye-promoted precipitation. The high sensitivity of the reaction offers the possibility of using this method for rapid concentration of very diluted protein solutions.     

Chromatography of proteins on columns of polyvinylpolypyrrolidone using adsorbed textile dyes as affinity ligands

A simple and inexpensive chromatography system for proteins is introduced. When the amino derivatives of chlorotriazine dyes or other azo dyes were added to an aqueous slurry of the crosslinked polymer polyvinylpolypyrrolidone they were adsorbed, thus forming an immobilized dye chromatographic matrix. The association of the textile dyes with polyvinylpolypyrrolidone did not prevent them from acting as affinity ligands for proteins. Parameters such as ionic strength, dye concentration, and column size modulated the affinity effect exerted by the immobilized dyes. Lysozyme present in an egg white protein mixture bound to a column onto which the amino derivative of Procion Brown H-A was adsorbed and was eluted with a linear gradient of KCl. The resulting purification of the enzyme was 37-fold with 80% of the original activity being recovered. Free dye eluting with the lysozyme was removed on a column of polyvinylpolypyrrolidone equilibrated with 0.5 M KCl. After chromatography, the dye column was regenerated with 0.5 M NaOH and recharged with dye. The system presented here allows one to initially screen large numbers of potentially useful protein ligands to optimize a protein separation, followed by scaleup to a system size determined by the user.     

Fractionation of proteins from human serum by counter-current distribution

Proteins of human serum have been fractionated by counter-current distribution using aqueous two-phase systems. These were composed of either polyethylene glycol and dextran or polyethylene glycol and the new water soluble starch polymer Aquaphase PPT. The distribution of serum proteins in the polyethylene glycol-Aquaphase PPT system resembles that in the polyethylene glycol-dextran system.The partition of a number of proteins could be changed by introducing polymer-bound reactive dyes into one of the phases. Due to affinity for the dyes several proteins were transferred into the phase containing the polymer-bound ligand leading to an improved separation of individual proteins.Furthermore, the effect of two different dyes, immobilised in the opposite phases, on counter-current distribution of serum proteins was demonstrated. The applicability of this method for fractionation of serum proteins is discussed.     

Dye-affinity techniques for bioprocessing: Recent developments

Textile or triazine dyes play an important role as affinity ligands in protein purification. Each step of the protein purification protocol can be divided into three stages, partitioning between two phases, separation of these phases and recovery of the target protein from the enriched phase. Now developments in dye-affinity techniques are discussed emphasizing the innovations in all three stages of the protein purification process. Dye-affinity chromatography has become a routine step in protein purification. New dyes have been developed and used successfully in both traditional chromatographic mode and new modes like affinity precipitation, polymer aqueous two-phase partitioning or expanded bed chromatography. The specificity of dye techniques has been increased by both purposeful designing of new dyes and decreasing non-specific protein–dye interactions with polymer shielding. One can envisage further development and ramification of dye-affinity techniuqes in protein purification.     

Protein blotting with direct blotting electrophoresis

Direct blotting electrophoresis, a method designed to be of general application for the separation and electroblotting of macromolecules, has been adapted to produce protein blots suitable for subsequent processing by standard techniques such as dye staining or immunological detection. After their separation in a very short gel the protein bands are electrophoresed out of the gel onto an immobilizing matrix. The matrix which is moved across the bottom of the gel by a conveyor belt binds these proteins with high affinity. Once the protein samples have been loaded onto the gel and electrophoresis has been started, no further intervention is needed until the blot is completed. The total expenditure of time for such a direct blot is less than 4 h for a mixture of proteins in the molecular weight range of 14-70 kDa. The staining sensitivity of directly blotted proteins is about 200 ng protein per band as revealed by India ink staining.     

Aqueous two-phase protein partitioning using textile dyes as affinity ligands.

A simple and inexpensive aqueous two-phase system for the affinity partitioning of proteins is introduced. An aqueous solution consisting of maltodextrin (M100; molecular mass, 1800) and polyvinylpyrrolidone (PVP360; molecular mass, 360,000) formed two phases at 4 degrees C when the concentration of the polymers was 22.5% (w/w) and 4.0% (w/w), respectively. When the amino derivatives of chlorotriazine textile dyes or other azo textile dyes were added to the two-phase system they partitioned asymmetrically, favoring the upper, less dense, PVP360-rich phase. The association of the textile dyes with PVP360 did not prevent them from acting as affinity ligands for proteins. Three of the dyes screened increased the partition coefficient of purified lysozyme nearly 50-fold over a control containing no dye. Parameters such as pH, ionic strength, and dye concentration modulated the affinity-partitioning effect of the system. The partition coefficient of lysozyme in an egg white protein mixture increased severalfold as the total protein content of the system approached 4% (w/w), indicating that protein concentration is also important in determining the partitioning characteristics of this two-phase system. Proteins were efficiently freed of PVP360 and textile dye by recovery in a high-salt solution when another two-phase system was formed upon the addition of a solution of concentrated potassium phosphate to the isolated upper phase of a PVP360/M100/textile dye two-phase system. The affinity-partitioning system presented here allows one to screen large numbers of potentially useful protein ligands to optimize protein separation, followed by direct scaleup to a system size determined by the user.     

Set of fluorochromophores in the wavelength range from 450 to 700 nm and suitable for labeling proteins and amino-modified DNA

We describe the synthesis, purification, and spectral properties of new dyes and reactive labels. They absorb in the visible range between 450 and 700 nm and display analytically useful fluorescence. They were made amino-reactive by esterification with N-hydroxysuccinimide (NHS). The resulting oxysuccinimide (OSI) esters were covalently linked to the amino groups of human serum albumin (HSA) or certain DNA oligomers. Except for dyes 9 and 13, they contain one reactive group only in order to avoid cross linking of biomolecules. Labeling of amino-modified biomolecules was performed by standard protocols, and the labeled proteins and oligonucleotides were separated from the unreacted dye by gel chromatography using Sephadex G25 as the stationary phase in the case of proteins, and reversed-phase HPLC in the case of DNA oligomers. The dyes also have been used as donor-acceptor pairs in fluorescence energy transfer systems and in energy transfer cascades.     

Highly fluorescent protein labeling using dendritic peptide derivatives.

Non-specific fluorescent dyes and photosensitizers are routinely used in clinical practice for the photodetection and photoablation of superficial lesions. Future applications in photomedicine are likely to rely on the selective delivery of photoactive compounds to diseased areas, using specific targeting agents such as antibodies. This fact underlines the need for methods that allow the chemically defined conjugation of several photoactive molecules to a single protein 'vehicle', with full retention of binding affinity. Here, we present methods for the site-specific fluorescent labeling of proteins using dendritic peptides, which had been chemically modified with multiple molecules of fluorescein. Branched peptide derivatives can be stably conjugated to proteins either by reaction with suitable free reactive groups or by using the high-affinity non-covalent interaction between calmodulin and a specific binding peptide. Chemical modification of proteins with one, two or four molecules of fluorescein resulted in a proportional increase in protein fluorescence.     

Dye-ligand affinity adsorbents for enzyme purification

Affinity chromatography is widely employed in laboratory and large-scale for the purification of biotherapeutics and diagnostics. Some of the most widely used ligands in affinity chromatography have been several reactive chlorotriazine dyes. In particular, immobilized anthraquinone dyes have found a plethora of applications in affinity chromatography because they are inexpensive, are resistant to chemical and biological degradation, are sterilizable and cleanable in situ, and are readily immobilized to generate affinity absorbents which display high binding capacity for a broad spectrum of proteins. This article provides detailed protocols on the preparation of a dye-ligand affinity adsorbent. Also, detailed protocols for effective application of these media, emphasizing binding and elution conditions are presented.     

Affinity chromatographic separation of secreted alkaline phosphatase and glucoamylase using reactive dyes

A single-step dye affinity chromatographic separation method was developed to separate secreted alkaline phosphatase (SEAP) and glucoamylase produced in CHO cell culture and Aspergillus niger fermentation, respectively. The reactive dye, Procion^® Green H-E4BD, was found to have a good binding capacity for SEAP, whereas Procion^® Blue H-ERD was the best dye ligand for glucoamylase. However, these dyes have a relatively low selectivity for the target protein. Consequently, elution of the adsorbed proteins by KCl solution resulted in a product with many impurity proteins as evident by the multiple protein bands on SDS-PAGE. However, elution of SEAP by its substrate, phosphate, produced a relatively pure protein with a high specific enzyme activity because of the competition for active site between the substrate and the dye ligand. Also, a high-purity glucoamylase product was obtained by elution with a borate solution. The relatively inexpensive dye affinity chromatography thus can be used for purifying enzymes from cell culture and fermentation broths. The adsorption of SEAP on the dye-ligand affinity resin followed the Langmuir isotherm. An axial dispersion model with external mass transfer limitation was developed to simulate the breakthrough curve in the chromatographic column. This mathematical model can be used to scale up the protein adsorption process.     

In vitro toxicity assays for dye ligands used in affinity chromatography

Some reactive textile dyes have been used for years as biomimetic ligands in protein purification. There has been reluctance, however, to use these dyes on a large scale for therapeutically applicable proteins for fear of possible dye leakage and consequent contamination. Therefore, toxicological data are necessary to quantify the level of this hazard. This study deals with a series of in vitro toxicity investigations with eukaryotic cells (growth, polyploidy, etc.) and with prokaryotic cells (Escherichia coli) for genotoxic studies. Both approaches demonstrated a lack of or slight toxicity for Reactive Blue 2 and Reactive Red 120 and their derivatives over the range 10–62.5 μg/ml in several assays.     

The applications of reactive dyes in enzyme and protein downstream processing

Downstream processing of proteins is often a key factor in the overall process of satisfying product specifications and meeting current commercial demands. In this context, affinity chromatography and other techniques based on the affinity concept have revolutionized protein purification technology, although they have failed to demonstrate their broader applicability at the process scale. On the other hand, reactive dyes offer many advantages as pseudoaffinity media and in many occasions have successfully circumvented problems associated with conventional affinity ligands. The main features of reactive dyes include their broad spectrum of interaction with proteins, low cost, ready availability, high reactivity, ease of immobilization, and both biological and chemical stability. Consequently, dye-ligand media now find application in both analytical and process-scale purification of proteins by techniques such as low- and high-pressure performance affinity chromatography, affinity partitioning, and affinity precipitation.     

Protein based microarrays: A tool for probing the proteome of cancer cells and tissues

A novel proteomic approach for probing cell and tissue proteome, which combines liquid phase protein separations with microarray technology has been developed. Proteins in cell and tissue lysates or in cellular subfractions are separated using any one of a number of separation modes which may consist of ion exchange liquid chromatography (LC), reverse phase LC, carrier ampholyte based separations, e.g. the use of Rotofor, affinity based separations, or gel based separations. Each first-dimension fraction obtained using one separation mode can be further resolved using one or more of the other separation modes to yield either purified protein in solution or liquid fractions with substantially reduced protein complexity. The advantage of a liquid based separation system is that proteins in hundreds of individual fractions can be arrayed directly and used as targets for a variety of probes. Constituent proteins in reactive fractions are identified by mass spectrometry and may be further resolved to determine the nature of the reactive protein(s). We present in this report initial data based on microarray analysis of individual Rotofor fractions obtained from lung adenocarcinoma cell line A549 lysates which have been probed with antibodies against specific proteins.     

A SNAP-tagged derivative of HIV-1--a versatile tool to study virus-cell interactions

Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches.Here we describe the construction and characterization of the HIV derivative HIV(SNAP), which carries the SNAP-tag as an additional domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIV(SNAP) represents a versatile tool which expands the possibilities for the analysis of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy.     

Pancreatic serine protease extraction by affinity partition using a free triazine dye

Affinity partitioning combines the partitioning behavior of biological macromolecules in aqueous two-phase systems with the principle of biorecognition. Among the numerous substances that have been evaluated as ligands, the reactive dyes constitute a group of low cost textile dyes which have proved to act as biomimetic ligands for many enzymes. The ability of reactive yellow 2 (RY2) to interact with trypsin (TRP) and chymotrypsin (ChTRP) and its behavior in aqueous two-phase systems formed by polyethylene glycol (PEG) and sodium citrate (NaCit) - were investigated. Different variables such as PEG molecular weight, tie line length and dye concentration were analyzed. RY2 showed to bind specifically to both TRP and ChTRP with affinity constants near to 10(3)M(-1). Its partition equilibrium is practically displaced to the top phase in systems formed by PEG of different molecular weight. Addition of this dye to PEG 8000/NaCit systems until a final concentration of 0.196% (w/w) induced an increase in TRP and ChTRP partition coefficients of at least 2 times over that in the absence of the ligand. These findings demonstrate that RY2 fulfils all the requirements to be considered as an affinity ligand in aqueous two-phase partitioning of TRP and ChTRP.     

Single-step affinity purification of recombinant proteins using a self-excising module from Neisseria meningitidis FrpC

Purification of recombinant proteins is often a challenging process involving several chromatographic steps that must be optimized for each target protein. Here, we developed a self-excising module allowing single-step affinity chromatography purification of untagged recombinant proteins. It consists of a 250-residue-long self-processing module of the Neisseria meningitidis FrpC protein with a C-terminal affinity tag. The N terminus of the module is fused to the C terminus of a target protein of interest. Upon binding of the fusion protein to an affinity matrix from cell lysate and washing out contaminating proteins, site-specific cleavage of the Asp-Pro bond linking the target protein to the self-excising module is induced by calcium ions. This results in the release of the target protein with only a single aspartic acid residue added at the C terminus, while the self-excising affinity module remains trapped on the affinity matrix. The system was successfully tested with several target proteins, including glutathione-S-transferase, maltose-binding protein, beta-galactosidase, chloramphenicol acetyltransferase, and adenylate cyclase, and two different affinity tags, chitin-binding domain or poly-His. Moreover, it was demonstrated that it can be applied as an alternative to two currently existing systems, based on the self-splicing intein of Saccharomyces cerevisiae and sortase A of Staphylococcus aureus.     

Affinity separation of proteins in aqueous three-phase systems

Aqueous polymer three-phase systems composed of dextran-Ficoll-polyethylene glycol-water have been used for affinity partition of proteins. The upper, middle, and lower phases are rich in polyethylene glycol, Ficoll, and dextran, respectively. Affinity partition was performed using the reactive dyes Cibacron Blue F36-A and Remazol Yellow GCL which are known as specific ligands for albumin and prealbumin from human serum. When the ligands were bound alternatively to polyethylene glycol, Ficoll, or dextran the target proteins were directed toward the upper, middle, or lower phase, respectively. In the presence of two ligands immobilized to two different polymers the distribution of two proteins could be steered to different phases at the same time. Serum albumin and prealbumin could be separated by using Cibacron Blue-Ficoll and Remazol Yellow-dextran or Cibacron Blue-polyethylene glycol and Remazol Yellow-dextran as polymer ligands.     

The structure and protein binding of amyloid-specific dye reagents

The self-assembling tendency and protein complexation capability of dyes related to Congo red and also some dyes of different structure were compared to explain the mechanism of Congo red binding and the reason for its specific affinity for beta-structure. Complexation with proteins was measured directly and expressed as the number of dye molecules bound to heat-aggregated IgG and to two light chains with different structural stability. Binding of dyes to rabbit antibodies was measured indirectly as the enhancement effect of the dye on immune complex formation. Self-assembling was tested using dynamic light scattering to measure the size of the supramolecular assemblies. In general the results show that the supramolecular form of a dye is the main factor determining its complexation capability. Dyes that in their compact supramolecular organization are ribbon-shaped may adhere to polypeptides of beta-conformation due to the architectural compatibility in this unique structural form. The optimal fit in complexation seems to depend on two contradictory factors involving, on the one hand, the compactness of the non-covalently stabilized supramolecular ligand, and the dynamic character producing its plasticity on the other. As a result, the highest protein binding capability is shown by dyes with a moderate self-assembling tendency, while those arranging into either very rigid or very unstable supramolecular entities are less able to bind.     

Symmetric and asymmetric squarylium dyes as noncovalent protein labels: a study by fluorimetry and capillary electrophoresis

Noncovalent interactions between two squarylium dyes and various model proteins have been explored. NN127 and SQ-3 are symmetric and asymmetric squarylium dyes, respectively, the fluorescence emissions of which have been shown to be enhanced upon complexation with proteins such as bovine serum albumin (BSA), human serum albumin (HSA), beta-lactoglobulin A, and trypsinogen. Although these dyes are poorly soluble in aqueous solution, they can be dissolved first in methanol followed by dilution with aqueous buffer without precipitation, and are then suitable for use as fluorescent labels in protein determination studies. The nature of interactions between these dyes and proteins was studied using a variety of buffer systems, and it was found that electrostatic interactions are involved but not dominant. Dye/protein stoichiometries in the noncovalent complexes were found to be 1:1 for SQ-3, although various possible stoichiometries were found for NN127 depending upon pH and protein. Association constants on the order of 10(5) and 10(7) were found for noncovalent complexes of SQ-3 and NN127, respectively, with HSA, indicating stronger interactions of the symmetric dye with proteins. Finally, HSA complexes with NN127 were determined by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). In particular, NN127 shows promise as a reagent capable of fluorescently labeling analyte proteins for analysis by CE-LIF without itself being significantly fluorescent under the aqueous solution conditions studied herein.     

Separation and detection methods for covalent drug-protein adducts

Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanism of toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug-protein adducts have important clinical and toxicological implications. In the last few decades, continuous progress in analytical methodology has been achieved with substantial increase in the number of new, more specific and more sensitive methods for drug-protein adducts. The methods used for drug-protein adduct studies include those for separation and for subsequent detection and identification. Various chromatographic (e.g., affinity chromatography, ion-exchange chromatography, and high-performance liquid chromatography) and electrophoretic techniques [e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and capillary electrophoresis], used alone or in combination, offer an opportunity to purify proteins adducted by reactive drug metabolites. Conventionally, mass spectrometric (MS), nuclear magnetic resonance, and immunological and radioisotope methods are used to detect and identify protein targets for reactive drug metabolites. However, these methods are labor-intensive, and have provided very limited sequence information on the target proteins adducted, and thus the identities of the protein targets are usually unknown. Moreover, the antibody-based methods are limited by the availability, quality, and specificity of antibodies to protein adducts, which greatly hindered the identification of specific protein targets of drugs and their clinical applications. Recently, the use of powerful MS technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight) together with analytical proteomics have enabled one to separate, identify unknown protein adducts, and establish the sequence context of specific adducts by offering the opportunity to search for adducts in proteomes containing a large number of proteins with protein adducts and unmodified proteins. The present review highlights the separation and detection technologies for drug-protein adducts, with an emphasis on methodology, advantages and limitations to these techniques. Furthermore, a brief discussion of the application of these techniques to individual drugs and their target proteins will be outlined.