Synchrotron radiation for direct analysis of metalloproteins on electrophoresis gels

Metalloproteomics requires analytical techniques able to assess and quantify the inorganic species in metalloproteins. The most widely used methods are hyphenated techniques, based on the coupling of a high resolution chromatographic method with a high sensitivity method for metal analysis in solution. An alternative approach is the use of methods for solid sample analysis, combining metalloprotein separation by gel electrophoresis and direct analysis of the gels. Direct methods are based on beam analysis, such as lasers, ion beams or synchrotron radiation beams. The aim of this review article is to present the main features of synchrotron radiation based methods and their applications for metalloprotein analysis directly on electrophoresis gels. Synchrotron radiation X-ray fluorescence has been successfully employed for sensitive metal identification, and X-ray absorption spectroscopy for metal local structure speciation in proteins. Synchrotron based methods will be compared to ion beam and mass spectrometry for direct analysis of metalloproteins in electrophoresis gels.     

Hydrophobic chromatography of β-galactosidase

The hydrophobic interaction of β-galactosidase with Sepharose 4B substituted with 3,3′-diaminodipropylamine was studied in both batch and column experiments. The equilibrium and the binding rate constants were determined for different phosphate buffer concentrations. The equilibrium constants exhibit a hysteresis effect, i.e., desorption constants are less than adsorption constants, and the higher the ionic strength to start the desorption, the larger the effect. The rate data are not satisfactorily described by a simple reversible first-order model. The column chromatographic data are semiquantitatively described by a local equilibrium theory without axial dispersion or intraparticle diffusion.     

Liposome/saline partition coefficients of low-molecular-weight solutes by gel chromatography

A chromatographic method for the determination of association constants of rapidly dissociable complexes is described and applied to quantification of liposome/saline partition coefficients using gel chromatography. The approach allows for estimation of the free solute concentration in the sample by simple manual processing of the intact right-hand part of the solute peak deformed due to gradual diffusion of the accumulated solute from the liposomes along the separation column. Validity of the procedure was confirmed by both reasonable agreement with equilibrium dialysis data and model-based deconvolution of the distorted peak into its two components corresponding to initially unbound compound and to that escaped from the liposomes during the separation process.     

Purification of human B cell growth factor

Human B cell growth factor (BCGF, 12,000 to 14,000 daltons) has been purified from lectin-stimulated, peripheral blood mononuclear cell-conditioned medium. The purification procedure involves a series of column chromatographic steps incorporating ion exchange, affinity binding, and gel filtration. This procedure is centered around a relatively high yield single chromatographic step, for the removal of co-eluting cytokines from BCGF, that is based on differential binding characteristics to the weak ion-exchange matrix, hydroxylapatite. Reverse-phase high-pressure liquid chromatographic separation on a C18-Bondapak column effectively separates the BCGF and TCGF moieties, yet is characterized by poor yields. High-pressure liquid chromatographic procedures on anion exchange and size exclusion provided the final purification step for BCGF, at an analytical level, resulting in a single band with a m.w. of 12,000 on a SDS-polyacrylamide gel.     

The elution profile of immobilized liposome chromatography: determination of association and dissociation rate constants

The interaction of lipophilic cations, tetraphenylphosphonium and triphenylphosphonium homologues with liposomes was investigated using immobilized liposome chromatography (ILC). Large unilamellar liposomes with a mean diameter of 100 nm were stably immobilized in chromatographic gel beads by avidin-biotin. The distribution coefficient calculated from (Ve-V0)/Vs (Ve, retention volume; V0, the void volume; Vs, the stationary phase volume) was found to be independent of flow rate, injection amount and gel bed volume, which is consistent with chromatograph theory. The relationship between the bandwidth and solvent flow rate did not follow band-broadening theories reported thus far. We hypothesized that the solvent might be forced to produce large eddies, spirals or turbulent flow due to the presence of liposomes fixed in the gel. Therefore, we developed a new theory for ILC elution: The column is composed of a number of thin disks containing liposomes and solution, and within each disk the solution is well mixed. This theory accounts for our results, and we were able to use it to estimate the rate constants of association and dissociation of the phosphonium to/from liposomes.     

On-column refolding of denatured lysozyme by the conjoint chromatography composed of SEC and immobilized recombinant DsbA

DsbA (disulfide bond formation protein A) located in the periplasm of Escherichia coli is a disulfide isomerase, which is vital to disulfide bonds formation directly affecting the nascent peptides folding to the correct conformation. In this paper, recombinant DsbA was firstly immobilized onto NHS-activated Sepharose Fast Flow gel. Then Sephadex G-100 gel was sequentially packed on the top of recDsbA Sepharose Fast Flow, and a so-called conjoint chromatography column composed of SEC and immobilized recombinant DsbA was constructed. Denatured lysozyme was applied on the conjoint column. The effect of SEC volume, flow rate, loading amount and volume, pre-equilibrium mode and KCl concentration in the buffer on lysozyme refolding were investigated in detail and the stability of DsbA immobilization was evaluated. Finally the reusability of the conjoint refolding column was also tested. When loading 2.4 mg denatured lysozyme in 0.5 ml solution, the activity recovery reached 92.7% at optimized experimental conditions, and the conjoint column renaturation capacity decreased only 7.7% after six run reuse due to the use of SEC section in the chromatographic refolding process. The conjoint chromatography offers an efficient strategy to refold proteins in vitro with high productivity and column reusability.     

Improved lectin-mediated immobilization of human red blood cells in superporous agarose beads

A new type of agarose bead, superporous agarose, was used as a gel support for immobilization of human red blood cells (RBCs) mediated by wheat germ lectin. The number of immobilized cells was similar to that obtained with commercial wheat germ lectin-agarose but the cell stability appeared to be superior. This allowed improved frontal affinity chromatographic analyses of cytochalasin B (CB)-binding to the glucose transporter GLUT1 which established a ratio of one CB-binding site per GLUT1 dimer for both plain RBCs or those treated with different poly amino acids. The measured dissociation constants, 70+/-14 nM for CB and 12+/-3 mM for glucose binding to GLUT1, are similar to those reported earlier.     

High-performance ion-exchange chromatographic separation of proteoglycans

Proteoglycans synthesized by cultured human muscle cells were separated by ion-exchange high-performance liquid chromatography using a Bio-gel TSK DEAE 5-PW analytical column. The procedure requires only 40 min to complete. The same analytical size column can be used for either analytical or semipreparative scale separations without significant loss of resolution. Proteoglycans elute from the TSK column with a similar recovery and at similar elution ionic strengths when compared to the established cellulose-based chromatographic gel, DEAE-Sephacel. The technique has been applied to the analysis of chondroitinase-digested samples and is particularly useful for rapid screening of large numbers of cultures for both biosynthetic rate studies and analysis of patterns of proteoglycan synthesis.     

Purification of liver aldehyde dehydrogenase by p-hydroxyacetophenone-sepharose affinity matrix and the coelution of chloramphenicol acetyl transferase from the same matrix with recombinantly expressed aldehyde dehydrogenase.

p-Hydroxyacetophenone was coupled to epoxy-activated Sepharose 6B to generate an affinity chromatographic matrix to purify aldehyde dehydrogenase. Purified beef liver mitochondrial aldehyde dehydrogenase specifically bound to the support and could be eluted with p-hydroxyacetophenone. A post-ammonium sulfate (30-55%) fraction of bovine liver was applied to the affinity gel column and aldehyde dehydrogenase was effectively purified, although not to complete homogeneity, indicating the potential selectivity of the matrix. Both beef liver cytosolic and mitochondrial aldehyde dehydrogenase bound to the column. A post-Cibacron blue Sepharose Cl-6B affinity-fractionated liver mitochondrial aldehyde dehydrogenase was purified to complete homogeneity by p-hydroxyacetophenone-Sepharose, thus eliminating the need for the isoelectric focusing step often employed. p-Hydroxyacetophenone was found to be a competitive inhibitor against propionaldehyde and noncompetitive against NAD. Escherichia coli lysates of recombinantly expressed aldehyde dehydrogenase were purified from E. coli lysates with one major 25-kDa protein contaminant also binding to the column, as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The 25-kDa contaminant was found to be chloramphenicol acetyl transferase from sequence analysis and binding studies.     

Analysis and Purification of Biological Stains by Gel Filtration

Some sixty biological stains of widely varying type have been subjected to gel filtration chromatography in columns of Sephadex LH-20 (Pharmacia, Uppsala) resin swollen by dimethylformamide saturated with NaCl. Most dyes contained more than one coloured constituent. Measures of their molecular weights were obtained. The use of the method for analysis was as effective as other common chromatographic or electrophoretic procedures but was technically simpler. Preparative use of the method merely involved using a larger column of gel, though occasionally use of ethanol as solvent gave better resolutions and did provide easier recovery of separated dyes.     

An estimation of the equivalent solution dielectric constant in the active-site cavity of metalloenzymes. Dependence of carboxylate-metal-ion complex stabilities on the polarity of mixed aqueous/organic solvents

The stability constants of the 1:1 complexes between Cu2+ and Zn2+ with formate, acetate and several phenylalkanecarboxylates, i.e. C6H5-(CH2)n-COO- with n = 0 to 5, are summarized for water, 50% aqueous ethanol and 50% aqueous dioxane (I = 0.1 M; 25 degrees C): Complex stability depends upon carboxylate group basicity. The influence of varying amounts of ethanol or dioxane (up to 90%) on the stability of the Cu2+ and Zn2+ (M2+) complexes with formate and acetate (CA) was measured by potentiometric pH titrations. The values for pKHH(CA) and log KMM(CA) increase, as expected, with increasing amounts of the organic solvents, i.e. with decreasing solvent polarity. The changes in the equilibrium constants are also evaluated with regard to the mole fractions of the organic solvents and the corresponding dielectric constants. These results may be used to estimate for low dielectric cavities in proteins the equivalent solution dielectric constant on the basis of enhanced carboxylate basicity or metal ion binding capability (method 1). Furthermore, the measured stability constants are used for comparisons of the coordination tendency of carboxylate ligands towards zinc(II)-metalloenzymes (method 2); in this way the equivalent solution dielectric constants in the active-site cavities of bovine carbonic anhydrase and carboxypeptidase A are estimated: the values are of the order of 35 and 70, respectively. This method seems to be generally applicable to metalloproteins.     

Simple radioassay for uridine phosphorylase and 5'-nucleotidase

A method is described whereby a large number of chromatographic fractions containing protein or enzyme components may be run on a single-starch gel. A multiple punch for making the sample holes, gel slab support, and a gel slicer used to cut several slices from the one gel for staining for the various enzyme or protien constituents are described. An example is given of a run using fractions obtained from a Sephadex G-200 column chromatography run of a herring muscle extract. Densitometry may be used to assay the stained gels. In another example a commercially obtained muscle lactate dehydrogenase preparation was chromatographed on a Sephadex G-200 column. The electrophoretic run of the fractions revealed the presence of both heart-type A and muscle-type B lactate dehydrogenase subunits and showed that the column run had effected a partial separation of the various tetramers.     

HPLC analysis of supernatant soluble factors from tumor cell variants with defined metastatic status

In the present study, we report a simple and sensitive procedure to study supernatant soluble factors of short time cultured cells by high performance gel permeation chromatography (HPGPC). This procedure enables a direct analysis with no preparatory procedures prior to application in the chromatographic column and eliminates tedious and often low reproducible techniques, required during conventional assay of culture medium. In addition, we show that HPGPC analysis is suitable for discriminating between two variants of a rhabdomyosarcoma with different metastatic potential, while two distinct cell lines (3LL carcinoma and B16F10 melanoma), which do not differ in their metastatic potential, do not present noticeable qualitative differences in their chromatographic secretion pattern.     

Determination of monoacetyldiamines and polyamines in urine by high-performance liquid chromatography

A procedure is described for the determination of monoacetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine in human urine. The procedure is based on the high-performance liquid chromatographic separation of the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivatives of these amines using two different chromatographic modes. Monoacetyl-1,6-diaminohexane was used as an internal standard. The amines were extracted from urine using a silica gel cartridge. The dansyl monoacetylpolyamines were separated from the mixture of dansyl derivatives of urinary amines on a bonded-phase CN column using a programmed solvent gradient elution. The dansyl acetylpolyamines were rechromatographed on a silica gel column.This chromatographic procedure was used for the determination of the concentration of N¹-acetylspermidine, N⁸-acetylspermidine and monoacetylputrescine in the urine of healthy volunteers and cancer patients.     

Purification of the beta subunit of the fibronectin receptor

In this work we describe a method for purification of the beta subunit of the mouse fibronectin receptor (GP135). Cellular glycoproteins were isolated from a detergent extract of SR-Balb tumor cell membranes by two steps of affinity chromatography on lentil lectin-Sepharose and wheat-germ-agglutinin--agarose. This material was subsequently bound to an Affi gel 102 column and eluted by increasing salt concentration. Most of the GP135 was eluted at 80 mM sodium chloride together with a few other components. A final step of hydroxyapatite chromatography in sodium dodecyl sulphate allowed elution of GP135 as a single chromatographic peak. Fractions containing GP135 were identified at each chromatographic step by immunoblotting with a specific antiserum. By this procedure GP135 was purified to homogeneity as judged by SDS-PAGE analysis of 125I-labelled material.     

Purification and properties of aldehyde dehydrogenase from Saccharomyces cerevisiae.

A procedure for the purification of aldehyde dehydrogenase from bakers' yeast (Saccharomyces cerevisiae) is reported. Treatment with acid, heat and organic solvents was avoided and chromatographic and filtration techniques in the presence of phenylmethylsulfonylfluoride were mainly used. An affinity chromatography step using the reactive dye Cibacron blue F3G-A, which was covalently bound to Sepharose 4B, was found to be essential. The enzyme was bound to and then released from the dye. The purified enzyme was shown to be homogeneous by gel filtration, disc electrophoresis and SDS electrophoresis. The molecular weight of the purified enzyme determined by gel filtration was 170,000, which agreed with that of the enzyme in the crude extract. The enzyme was composed of subunits of a molecular weight of 57,000. The specific activity of the enzyme was 20 units per mg of protein under the standard assay conditions. The substrate specificity, the relative maximal velocity, the michaelis constants, the pH optimum, the stability and the activation energy of the enzyme are reported.     

Functional role of metalloproteins in genome stability

Cells contain a large number of metalloproteins that commonly harbor at least one metal ion cofactor. In metalloproteins, metal ions are usually coordinated by oxygen, sulfur, or nitrogen centers belonging to amino acid residues in the protein. The presence of the metal ion in metalloproteins allows them to take part in diverse biological processes, such as genome stability, metabolic catalysis, and cell cycle progression. Clinically, alteration of the function of metalloproteins in mammals is genetically associated with diseases characterized by DNA damage and repair defects. The present review focuses on the current perspectives of metal ion homeostasis in different organisms and summarizes the most recent understanding on magnesium, copper, iron, and manganese-containing proteins and their functional involvement in the maintenance of genome stability.     

Stability assessment of different chelating moieties used for elemental labeling of bio-molecules

Integrating elemental labeling in quantitative LC-ICP-MS based bio-analysis requires fundamental experiments concerning the stability of complexes during analysis. In a competitive approach complex stability of the chelating moieties 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraaceticacid (DOTA), 1,4,7-triazacyclononane-N,N',N'-triacetic acid (NOTA) and diethylenetriaminepentaacetic dianhydride (DTPA) in combination with 11 different lanthanides was investigated under typical chromatographic conditions. Measurements were carried out via LC-ICP-QMS using a novel mixed mode separation method. The influence of chromatographic separation, pH and temperature on complex stability constants was assessed regarding further applications of multiplexing in bio-analytical assays. The limit of detection (LOD) for LC-ICP-QMS was 0.03 nM for all investigated Tm complexes (0.15 fmol absolute). Quantification of the complexes was performed via external, flow injection based calibration. For all investigated complexes the stability was significantly decreased by the chromatographic conditions. Moreover, complexation by DOTA revealed two different signals suggesting the presence of a stable intermediate product. Ln(3+)-DOTA and Ln(3+)-NOTA complexes provided high stability at 5 °C and 37 °C over a time of 12 hours, whereas Ln(3+)-DTPA complexes showed significant degradation at 37 °C.     

Analysis of phase composition in aqueous two-phase systems using a two-column chromatographic method: Application to lactic acid production by extractive fermentation

A new chromatographic system for the simultaneous analysis of polyethylene glycol, dextran, sugars, and low-molecular-weight fatty acids was developed. The system is based on a gel exclusion column which allows a first separation between high- and low-molecular-weight compounds, and a cationic exchange column used to further separate the low-molecular-weight compounds. Two applications of the system were demonstrated: (i) after optimizing eluent conditions the gel exclusion column was used to determine the influence of lactic acid, phosphate buffer, and lactic acid bacteria on the ethylene oxide propylene oxide-dextran T40 phase diagram by HPLC; (ii) the ion exchange column was coupled in series with the gel exclusion column and the concentration of polyethylene glycol, dextran, glucose, lactate, acetate, and formate was determined in samples from the fermentative production of lactic acid in a polyethylene glycol 8000-dextran T40 aqueous two-phase system. The fermentation was operated without pH control in a repeated extractive batch mode, where the cell-free top phase was replaced four times, whereas the cell-containing bottom phase was reused repeatedly. The yield was 1.1 mol of lactic acid formed per mole of glucose added and the productivity was 4.7 mM.h(-1). The polymeric composition of the fermentation system was monitored during the five repeated extractive batches, and it showed a progressive depletion in polyethylene glycol and a progressive enrichment in dextran. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 303-311, 1997.     

Characterization of the platelet aggregation inducer and inhibitor isolated from Crocus sativus

Bulbs of Crocus sativus variety Cartwrightianus were found to contain both a platelet aggregation inducer and inhibitor. The aggregating factor has a Mr of 42 kDa estimated by a Sephadex G75 column and SDS-polyacrylamide slab gel electrophoresis. It was found to lack enzymatic activity such as proteinase, esterase and acid or alkaline phosphatase. The inhibitory factor was also purified to homogeneity by different chromatographic techniques and shows a Mr of 27 kDa as it was estimated by Biogel P30 column and SDS-polyacrylamide slab gel electrophoresis. It was found to possess strong proteinase activity.