Chromatographic and electrophoretic methods for modified hemoglobins

The discovery of the clinically important glycohemoglobin adducts and their relation to diabetes mellitus have greatly stimulated the study of other minor post-translational modifications of hemoglobin. Chromatographic and electrophoretic procedures have played an important role in these studies. Today several hemoglobin adducts are known and the formation of adducts with glucose, phosphorylated carbohydrates, urea/cyanate, aspirin, vitamins, acetaldehyde, penicillin and acetyl CoA have been described. Furthermore, new adducts, such as those observed using hemoglobin as a biochemical marker monitoring environmental, occupational and lifestyle exposures to reactive toxic chemicals are constantly being reported. This review deals with chromatographic and electrophoretic separation methods available for the study of non-enzymatic post-translational modifications of hemoglobin. Suitability, perspectives and biomedical applications are discussed.     

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.     

Chromatographic and electrophoretic methods related to the carbonic anhydrase isozymes

There are three gene families that encode zinc metalloenzymes that catalyze the reversible hydration of CO2. The encoded enzymes are termed carbonic anhydrases (CAs). The CA isozymes have been purified from representatives of all types of organisms. Most CAs are strongly inhibited by aromatic sulfonamides. Several chromatographic and electrophoretic methods have been devised to determine binding constants for sulfonamides to CAs, and these compounds have been extensively used for, often single-step, affinity chromatographic separation of CAs from complex matrixes. The purification of different CA isozymes from different organisms is reviewed, as are methods for detection of CAs during chromatography and electrophoresis.     

Carboxylic acids: prediction of retention data from chromatographic and electrophoretic behaviours

A review of the main results reached in the prediction of retention data of carboxylic acids, inferred by their chromatographic and electrophoretic behaviour, is presented. Attention has been focused on the main separation methods used in carboxylic acids analysis, that is ion-exclusion, anion-exchange, reversed-phase (RP) liquid chromatography and capillary electrophoresis. Papers proposing mechanistic models as well as chemometric and multilayer feed-forward neural network analysis of ion chromatography (IC) and RP chromatographic retention data were reviewed. Principal component analysis, PCA, sequential simplex method and simultaneous modelling of response surfaces through simple nonlinear models (not related to equilibria involved in retention) have been considered. Computer simulations for the prediction of retention data have also been discussed. A quick overlook on the prediction of capacity factors of analytes by less common determination methods such as thin-layer, gas chromatography and supercritical fluid chromatography has also been done.     

Separation methods for glycoprotein analysis and preparation

Several chromatographic methods have been developed for the isolation and characterization of glycoproteins. In these methods, affinity chromatography, a single-step method, or combined use with general chromatographic methods have now become essential for the purification of many biologically important glycoproteins, including alpha1-acid glycoprotein, immunoglobulins, ceruloplasmin and erythropoietin. On the other hand, almost all glycoproteins exhibit polymorphism associated with their glycan moieties. This feature is wide-spread and has been observed in natural as well as in recombinant DNA glycoproteins. Recently, several sophisticated techniques--such as electromigration method (high-performance capillary electrophoresis) and chromatographic methods (two-dimensional polyacrylamide gel electrophoresis, high-pH anion-exchange chromatography with pulsed-amperometric detection)--have been introduced for qualitative or quantitative estimation of the microheterogeneity of glycoproteins. For gaining further insight into the structure-function relations for microheterogeneity, preparative chromatographic techniques that can yield sufficient quantities of glycoprotein variants must be developed.     

Separation of 24 dansylamino acids by capillary electrophoresis with a non-ionic surfactant

The separation of 24 dansylamino acids was investigated by capillary electrophoresis with an additive of micelles of a non-ionic surfactant, Tween 20. Although two pairs of peaks, norvaline and methionine derivatives, and didansyltyrosine and solvent (methanol), did not show good resolution, other dansylamino acids were well separated within 70 min using 100 mM Tween 20 and pH 2.40. The theoretical plate numbers calculated for dansylamino acids were 28,000-111,000 with a 19-cm capillary column.     

Efficient methods of processing metal chips

The development and use of efficient methods of processing chips (particularly alloy chips) is important for solving the problems of conserving resources, improving quality of the metal that is produced, and improving the environmental situation at metallurgical plants.     

Clinical analysis of human urinary proteins using high resolution electrophoretic methods

The application of isoelectric focusing (IEF), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional electrophoresis (2-DE) and capillary electrophoresis (CE) for high resolution electrophoretic analysis of human urinary proteins is reviewed. In each case, the information is tabulated chronologically with details of sample preparation, electrophoretic system, detection method and clinical application. The text includes an historical perspective of the use of each method for urinalysis and a detailed review of the application of the methods to the investigation of renal disease, renal transplantation, Bence Jones proteinuria (BJP), diabetes mellitus, cadmium toxicity, nephrolithiasis and cancers of the urogenital tract.     

Separation and characterization of microtubule proteins from calf brain

Electrophoresis of microtubule preparations purified from calf brain by repeated cycles of assembly and disassembly shows that they contain many proteins in addition to alpha- and beta-tubulin. These additional proteins constitute about 17% of the total material present after five cycles of assembly and disassembly. Both one-dimensional and two-dimensional (P.H. O'Farrell (1975), J. Biol. Chem. 250, 4007) electrophoretic techniques have been used to characterize them. They can be divided into two groups: one that contains proteins which remain in constant quantitative ratio to tubulin during the purification cycles, and one composed of proteins which are removed during purification, although inefficiently. Gel-filtration chromatography of cold-depolymerized microtubule preparations yields a polydisperse fraction of high molecular weight containing most of the non-tubulin proteins. This fraction contains flexible filaments about 100 A in diameter similar to those reported by R.A.B. Keats and R.H. Hall ((1975), Nature (London) 247, 418). It is suggested that these fibers are neurofilaments, and that they may be the major source of the group of inefficiently removed proteins.     

Organic halogen removal from chlorinated humic ground water and lake water by nitrifying fluidized-bed biomass characterised by electron microscopy and molecular methods

The dechlorinating and genotoxicity-removing activities of nitrifying fluidized-bed reactor biomass towards chlorinated organic compounds in water were shown at level below 1 ppm. The removal rates of adsorbable organic halogens were 200 micrograms Cl (g VS day)-1 for chlorinated humic ground water and 50 micrograms Cl (g VS day)-1 for chlorinated lake water when studied in batch mode. In a sequenced batch mode the removal rates [micrograms Cl (g VS day)-1] were 2000 from chlorohumus, 1400-1800 from chlorophenols in chlorinated ground water, and 430-720 from chlorohumus in chlorinated lake water. Genotoxicity was removed to a large extent (60%-80%) from the chlorinated waters upon incubation with nitrifying reactor biomass. 2,6-Di-, 2,4,6-tri and 2,3,4,6-tetrachlorophenols competed with chlorinated water organohalogens for dechlorination. The dechlorination of chlorophenols and chlorohumus required no ammonia and was not prevented by inhibitors of ammonia oxidation, nitrapyrin, parathion, sodium diethyldithiocarbamate, or allylthiourea. Electron microscopical inspection of the biomass showed the dominance of clusters of bacteria resembling known nitrifying species, Nitrosomonas, Nitrobacter, and Nitrosospira. This was supported by polymerase chain reaction amplification of the biomass DNA with four different primers, revealing the presence of 16S rDNA sequences assignable to the same species. The most intensive band obtained with the Nitroso4E primer was shown to be closely related to Nitrosomonas europaea by restriction analysis.     

The use of electrophoretic methods for determining modified proteins in diabetic patients

Modified proteins were determined by isoelectric focusing in borate-polyol system with subsequent colorimetry (micromethod) and electrophoresis of blood serum on paper with subsequent TCA-ethanol treatment. Increased levels of glycated hemoglobin and modified albumin and changed light absorbance of glycated albumin were detected. The levels of glycated hemoglobin assessed by the micromethod and colorimetry without calibration did not correlate.     

Separation of aromatic acids, DOPA, and methyl-DOPA by capillary electrophoresis with dendrimers as buffer additives

The underlying disease of a candidate for lung transplantation, especially advanced pulmonary fibrosis, can cause particular and dramatic difficulties. Pulmonary fibrosis is the end-result of a variety of pathological diseases and their associated processes. This article summarizes the diagnosis and management of some of the more common causes of fibrosis, outlines their natural histories and treatment outcomes, and describes the trade-off of pulmonary fibrosis for lung transplantation. Four main categories of end-stage fibrosis are discussed: idiopathic pulmonary fibrosis, sarcoidosis, pulmonary fibrosis from systemic diseases or drugs, and occupational- or environmental-related pulmonary fibrosis. Each group will be covered systematically and the options and indications for lung transplantation will be addressed.