Chromatographic methods for amylases

This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of α- and β-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.     

Chromatographic and electrophoretic methods for analysis of superoxide dismutases

A brief overview of the family of superoxide dismutase (SOD) enzymes and their biomedical significance is presented. Methodology for the purification and electrophoretic analysis of superoxide dismutases is reviewed and discussed, with emphasis on the specific problems raised by the separation of individual superoxide dismutase isoenzymes. Purification methods and their performance, as reported in the literature, are summarised in table form. Generally used methods for measuring SOD activity in vitro and SOD visualisation after electrophoresis are outlined, particularly those relevant to the monitoring of progress of SOD purification.     

Kallikrein and kallikrein-like proteinases: purification and determination by chromatographic and electrophoretic methods

Kallikreins and kallikrein-like enzymes make up a family of serine proteinases present in tissues and body fluids of mammals and in some snake venoms. This review deals with the procedures of purification, detection and determination of these enzymes by chromatographic and electrophoretic methods. The procedures are reported in tables, described and discussed with the aim of illustrating the state-of-the-art of research in the field.     

Chromatography of hydroxysteroid dehydrogenases

The structure-function study of hydroxysteroid dehydrogenases has stimulated the development of their chromatography, which in turn reveals more mechanisms of these enzumes. Due to the various membrane associations and mild hydrophobic nature of most of the enzymes studied up to now, hydrophobic interaction chromatography has played a crucial role in their purification, using media such as phenyl-Superose or Sepharose-PEG. At the same time, affinity chromatography, especially the dye-containing columns, proves very efficient for these dehydrogenases, as the latter utilizes adenylyl-containing cofactors. Elution by their specific ligand facilitates their purification. In this paper, the use of detergents in the purification of these enzymes is also reviewed. Hydroxysteroid dehydrogenase preparation is further improved by rapid purification which facilitates the elimination of protein microheterogeneity, caused in vitro by oxidation, reduction or partial proteolysis. This process was shown to increase the crystallizability of the enzymes [Lin et al., J. Cryst. Growth, 122 (1992) 242–245; Zhu et al., J. Mol. Biol., 234 (1993) 242–244]. The fast purification permitted a simpler procedure and better combination of various columns than conventional chromatography. This leads to even higher efficiency, yielding homogeneous and highly active preparations.     

Purification and characterization of an aminopeptidase from the chloroplast stroma of barley leaves by chromatographic and electrophoretic methods

Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of protein or peptide substrates. Although some aminopeptidase activities have been found in plant chloroplasts, the identity of these proteins remains unclear. In this work, we report the purification to apparent homogeneity of a soluble aminopeptidase from isolated barley chloroplasts which preferentially degraded alanyl-p-nitroanilide (Ala-pNA). After organelle isolation in a density gradient and precipitation of soluble proteins with ammonium sulfate, the proteins were purified in three consecutive steps including hydrophobic interaction, gel permeation and ion-exchange chromatographies. The purified enzyme appeared as a single band with a M_r of 84 000 in sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The M_r of the native enzyme was estimated to be 93 000 by gel permeation chromatography, suggesting that the protein is a monomer. Mass spectrometry analysis of tryptic digests indicates that the primary structure of the protein has not been reported previously. The enzyme was characterized as a metalloprotease as it could be totally inhibited by 1,10-phenanthroline. Strong inhibition could also be observed using the specific aminopeptidase inhibitors amastatin and bestatin. Besides Ala-pNA, the purified protein could also cleave with decreasing activity glycyl-pNA, leucyl-pNA, lysyl-pNA, methionyl-pNA and arginyl-pNA. The possible physiological role of this enzyme in the chloroplast stroma is discussed.     

Tyrosine protein kinase assays

Protein kinases form a large family of enzymes that play a major role in a number of live processes. The study of their action is important for the understanding of the transformation mechanisms and of the normal and pathological growth events. The quality of an enzyme assay is often the key point of an enzymatic study. It must be flexible and compatible with various experimental conditions, such as those for the purification process, the screening of inhibitors and the substrate specificity studies. As will be shown in the present review, two categories of substrates, peptidic and proteic, should be distinguished. The use of peptide substrates facilitates the determination of the recognition requirements of the enzyme and of the kinetic effects of even minute variations in their sequence. These linear peptide structures are assumed to mimic a complex interaction between the enzyme and a proteic substrate in which distant amino acids in the sequence are vicinal in the folded substrate. Less amenable to a systematic study, but probably more adequate to investigate the natural substrate of a given kinase, are the proteic substrates. Obviously the tools to measure protein kinase activities are not the same in these two cases. The main difficulty in assaying protein kinases is the use of labelles γ-ATP, mostly at large excess concentration, since the final product of the reaction has to be separated from the non-reacted labelled ATP. In the case of peptide substrates, the difficulty is to separate them from ATP basing on differences of molecular mass. Despite the efforts of many investigators to rely upon differences in solubility, in charges or in “affinity”, this separation, which is crucial for the assay, is still an unsolved experimental problem. Chromatographic, as well as electrophoretic assays appeared relatively late in this domain, and more work in assessing new methodologies might bring new breakthroughs in the next few years. Specific, simple and reliable kinase assays are still a major challenge. Their improvement will help to conduct specificity studies, to elucidate complex growth mechanisms in which they are involved and to discover more selective potent inihibitors.     

Separation and functional analysis of eukaryotic DNA topoisomerases by chromatography and electrophoresis

DNA topoisomerases are enzymes that control DNA topology by cleaving and rejoining DNA strands and passing other DNA strands through the transient gaps. Consequently, these enzymes play a crucial role in the regulation of the physiological function of the genome. Beyond their normal functions, topoisomerases are important cellular targets in the treatment of human cancers. In this review we summarize current protocols for extracting and purifying DNA topoisomerases, and for separating subtypes and isoforms of these enzymes. Furthermore, we discuss methods for measuring the catalytic activity of topoisomerases and for monitoring the molecular effects of topoisomerase-directed antitumor drugs in cell-free assays.     

Purification of a -hydantoinase using a laboratory-scale Streamline phenyl column as the initial step

A -hydantoinase from Thermus sp. was overexpressed in Escherichia coli and purified to homogeneity for subsequent crystallization. The purification was performed with hydrophobic interaction chromatography as the capture step followed by anion-exchange chromatography and gel permeation chromatography as intermediate purification and polishing steps, respectively. The hydrophobic interaction step was done in fluidized bed mode in a laboratory-scale Streamline column made from conventional laboratory equipment. The whole purification protocol could be finished within one day. The purified enzyme crystallizes. The crystals are suitable for X-ray protein structure analysis and diffract to at least 2.3 Å resolution. Complete data sets have been measured up to 2.6 Å resolution. The X-ray structure is currently being solved.     

Purification of cytochrome P-450 from adult pig testis by hydroxylapatite and deoxycorticosterone affinity column chromatography

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and deoxycorticosterone affinity column chromatography. Cytochrome P-450 was determined to have an isoelectric point of 6.5 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular mass was estimated to be 52 000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A 1000-fold purification was achieved with a yield of 5%.     

Purification of acetohydroxy acid synthase by separation in an aqueous two-phase system

Extraction in a polyethylene glycol (PEG)–phosphate aqueous two-phase system was considered as a primary step in purification of the acetohydroxy acid synthase III large catalytic subunit from an E. coli extract. Extraction optimization was achieved by varying the system parameters. Two systems with the following weight compositions were chosen for purification: PEG-2000 (16%)–phosphate (6%) and PEG-4000 (14%)–phosphate (5.5%)–KCl (8%), both at pH 7.0 and 1 mg total protein per 1 g system. Significant purification was achieved by a single extraction step with 70% recovery of the enzyme. After an additional ion-exchange chromatography step, pure enzyme was obtained in a 50% overall yield.     

Purification of the fengycin synthetase multienzyme system from Bacillus subtilis b213

The purification of the multienzyme system producing the lipodecapeptide fengycin in Bacillus subtilis b213 was investigated. By gel filtration of a cell free extract of this organism three enzyme fractions were obtained from which five multifunctional components of fengycin synthetase were separated by high resolution anion-exchange FPLC procedures. These proteins were characterized by their thioester formation activities with ¹⁴C-labeled substrate amino acids and by N-terminal sequencing. Correlation of these data with the DNA sequences of the pps (fen) operons in three B. subtilis strains provided detailed knowledge on the structural and functional organization of fengycin synthetase.     

Essential role of the concentration of immobilized ligands in affinity chromatography:: Purification of guanidinobenzoatase on an ionized ligand

Guanidinobenzoatase, a plasma protein with possible application as a ‘tumor marker’, has been fully purified by one-step affinity chromatography. The affinity matrix was prepared by ‘controlled’ immobilization of an enzyme inhibitor (agmatine) onto commercial agarose gels containing carboxyl moieties activated as N-hydroxysuccinimide esters. In this way, agmatine becomes immobilized through an amido bond and preserves an ionized guanidino moiety. Different matrices with different concentration of ligands were prepared in order to evaluate their properties as affinity supports. Interestingly, matrices with a very low concentration of immobilized ligands (2 μmol/ml, corresponding to the modification of only 5% of active groups in the commercial resins) exhibited a low capacity for unspecific adsorption of proteins (as anion-exchange resins) and displayed also a high capacity for specific adsorption of our target protein. On the other hand, when affinity matrices possessed a moderate concentration of agmatine (10 μmol/ml of gel or higher), two undesirable phenomena were observed: (a) the matrix behaves as a very good anionic exchange support able to non-specifically adsorb most of plasma proteins and (b) the specific adsorption of our target protein becomes much lower. The latter phenomenon could be due to steric hindrances promoted by the interaction between each individual immobilized ligand and the corresponding binding pocket in the target protein. These hindrances could also be promoted by the presence of a fairly dense layer of immobilized ligands covering the support surface, thus preventing interactions between immobilized ligands and partially buried protein-binding pockets. In this way, a successful affinity purification (23.5% yield, ×220 purification factor, a unique electrophoretic band) could be achieved by combination of three approaches: (i) the use of affinity matrices possessing a very low density of immobilized ligands, (ii) performing affinity adsorption at high ionic strength and (iii) performing specific desorption with substrates or substrate analogues.     

Purification of angiotensin I converting enzyme from pig lung using concanavalin-A sepharose chromatography

Angiotensin I converting enzyme (ACE) plays a major role in blood pressure regulation, catalyzing the conversion of angiotensin I to the vasoconstrictor angiotensin II. In this report we describe a two-step affinity chromatography method for preparative purification of ACE from pig lung using Concanavalin-A Sepharose 4B and affinity chromatography on Lisinopril Sepharose 6B. The same purification scheme was used to obtain Cobalt-ACE, where zinc ion located at the active site is replaced by cobalt. Cobalt-ACE visible spectrum shows a characteristic broad peak from 500 to 600 nm. The shape and maximum absorptivity of this peak changes in presence of ACE inhibitors that bind at the catalytic site.     

High-performance liquid chromatographic methods for the determination of topoisomerase II inhibitors

Various methods for separating eleven different types of topoisomerase II (TOPO-2) inhibitors, including epipodophyllotoxins, anthracyclines, anthracenediones, anthrapyrazoles, anthracenebishydrazones, indole derivatives, aminoacridines, benzisoquinolinediones, isoflavones, bisdioxopiperazines and thiobarbituric acids, are summarized. Proper sample preparation and storage is critical to the successful analysis of some TOPO-2 inhibitors due to difficulties associated with adsorption, instability and complex biological components. Solid-phase and liquid–liquid extractions are widely used to separate TOPO-2 inhibitors from biological samples, although simple deproteinization followed by direct analysis of the supernatant is preferable to extraction based on its speed and simplicity. High-performance liquid chromatography (HPLC) is the favored method for the bioanalysis of TOPO-2 inhibitors. UV or diode array detection is generally employed for early pharmacokinetic studies, while fluorescence or electrochemical detection is used more frequently for analytes with fluorescent or oxidative–reductive properties. For analyses requiring highly sensitive and/or specific detection, electrospray mass spectrometry (ESI-MS or ESI-MS–MS) provides a suitable alternative. A comprehensive compilation of the HPLC techniques currently used to separate TOPO-2 inhibitors will aid the future development of analytical methods for new TOPO-2 inhibitors.     

Recent advances in chromatographic and electrophoretic methods for the study of drug-protein interactions

Drug-protein binding is an important process in determining the activity and fate of a pharmaceutical agent once it has entered the body. This review examines various chromatographic and electrophoretic methods that have been developed to study such interactions. An overview of each technique is presented along with a discussion of its strengths, weaknesses and potential applications. Formats that are discussed include the use of both soluble and immobilized drugs or proteins, and approaches based on zonal elution, frontal analysis or vacancy peak measurements. Furthermore, examples are provided that illustrate the use of these methods in determining the overall extent of drug-protein binding, in examining the displacement of a drug by other agents and in measuring the equilibrium or rate constants for drug-protein interactions. Examples are also given demonstrating how the same methods, particularly when used in high-performance liquid chromatography or capillary electrophoresis systems, can be employed as rapid screening tools for investigating the binding of different forms of a chiral drug to a protein or the binding of different proteins and peptides to a given pharmaceutical agent.     

Antitumor drugs possessing topoisomerase I inhibition: applicable separation methods

Separation methods for antitumor drugs capable of topoisomerase I inhibition were reviewed in this study. Camptothecin (CPT) its related analogues seemed to be promising anticancer drugs that exhibit topoisomerase I inhibition. This group of compounds contain a closed α-hydroxy-δ-lactone ring (lactone form) that can undergo reversible hydrolysis to form the open-ring form (carboxylate form). In vitro pharmacological study showed that the antitumor activity of the lactone form was higher than that of the carboxylate form. Thus a quantitative method to separate these two forms is important to evaluate the pharmacokinetics and pharmacodynamics of these compounds. Nevertheless, current separation methods are complicated by the pH-dependent instability of the lactone moiety. High-performance liquid chromatography (HPLC) coupled with fluorometric detection has been widely used for the quantitation of the drug as the intact lactone form or as the total lactone carboxylate forms in biological matrices. In this report we reviewed current applicable chromatographic techniques for further bioanalytical studies of CPT derivatives including sample preparations, HPLC columns, mobile phases and additives.     

Purification of penicillin G acylase using immobilized metal affinity membranes

The immobilized metal affinity membrane (IMAM) with modified regeneration cellulose was employed for purification of penicillin G acylase (PGA). For studying PGA adsorption capacity on the IMAM, factors such as chelator surface density, chelating metal, loading temperature, pH, NaCl concentration and elution solutions were investigated. The optimal loading conditions were found at 4 degrees C, 0.5 M NaCl, 32.04 micromol Cu(2+) per disk with 10 mM sodium phosphate buffer, pH 8.5, whereas elution conditions were: 1 M NH(4)Cl with 10 mM sodium phosphate buffer, pH 6.8. By applying these chromatographic conditions to the flow experiments in a cartridge, a 9.11-fold purification in specific activity with 90.25% recovery for PGA purification was obtained. Meanwhile, more than eight-times reusability of the membrane was achieved with the EDTA regeneration solutions.     

O-Methylation of L-dopa in Melanin Metabolism and the Presence of Catechol-O-Methyltransferase in Melanocytes

O-Methylation of L-dopa was investigated as a possible regulatory mechanism in melanin metabolism. The methylation product of L-dopa, 3-O-methoxytvrosine was detected in extracts of cultured human melanocytes. The enzyme catechol-O-methyltransferase is responsible for this O-methylation and that of the dihydroxyindolic intermediates of melanogenesis. The enzyme is present in melanocytes in its soluble and membrane-bound isoforms. Immuno-electron microscopy suggests the presence of the membrane-bound enzyme in the endoplasmic reticulum. This localization may indicate a role of catechol-O-methyltransferase in protecting the melanocyte against reactive dihydroxyphenolic intermediates of melanogenesis leaking from the melanogenic compartments. On the other hand, the O-methylation of L-dopa may serve as a regulatory point in melanogenesis during early stage of tyrosinase processing in the endoplasmic reticulum.     

Purification of uricase from mammalian tissue.

A simple, rapid procedure for the purification of uricase from mammalian tissue is reported. The procedure is based on the precipitation of mammalian uricase under certain dialysis conditions, and on its low solubility near neutral pH. Exceptionally high yields of homogeneous enzyme are obtained.     

Purification and biochemical characterization of two extracellular peroxidases from Phanerochaete chrysosporium responsible for lignin biodegradation

Two extracellular peroxidases from Phanerochaete chrysosporium, namely a lignin peroxidase (LiP) and manganese peroxidase (MnP), were purified simultaneously by applying successively, ultrafiltration, ion-exchange and gel filtration chromatography. LiP and MnP have a molecular mass of 36 and 45 kDa, respectively. The optimal pHs for LiP and MnP activities were 3.0 and 4.5, respectively. Both peroxidases showed maximal activity at 30 °C and moderate thermostability. MnP activity was strongly inhibited by Fe²⁺, Zn²⁺, Mg²⁺ and Hg²⁺, and enhanced by Mn²⁺, Ca²⁺ and Cu²⁺. LiP activity was enhanced by Ca²⁺, Na⁺ and Co²⁺ and it was inhibited in the presence of K⁺, Hg⁺, Fe²⁺, Mg²⁺ and high concentrations of Cu²⁺ and Zn²⁺. The K_m and V_max for LiP toward veratryl alcohol as a substrate were 0.10 mM and 15.2 U mg⁻¹, respectively and for MnP toward Mn²⁺, they were respectively 0.03 mM and 25.5 U mg⁻¹. The two peroxidases were also able to break down rice lignin in a small-scale solid state treatment system. Data suggest these two peroxidases may be considered as potential candidates for the development of enzyme-based technologies for lignin degradation.