Fractionation and isolation of the multiple forms of prorennin (prochymosin)

The heterogeneity of prorennin was studied by chromatography on DEAE-cellulose and microgranular DEAE-cellulose columns, as well as by polyacrylamide-gel electrophoresis. Prorennin prepared by alum treatment, salting-out and chromatography was resolved into three components by a compound gradient of sodium phosphate on microgranular DEAE-cellulose. Polyacrylamide-gel electrophoresis confirmed the chromatographic results, but crystalline rennin was shown to consist of four bands. When prorennin was isolated directly by chromatography, four zymogen components were resolved on microgranular DEAE-cellulose with a modified compound gradient of sodium phosphate. Polyacrylamide-gel electrophoresis confirmed the existence of four multiple forms of prorennin as well as homogeneity of the chromatographic fractions.     

Gradient-thickness thin-layer chromatography for the isolation and analysis of trace amounts of free fatty acids in large lipid samples

A thin-layer chromatographic method for quantitative isolation of free fatty acids is described. This method appears to be more satisfactory than existing methods in offering the combination of advantages of specificity, simplicity, rapidity, reproducibility, accuracy, high sensitivity, and applicability as a preparative technique. The method involves chromatography on a thin-layer plate on which the layer of Silica Gel G decreases linearly in thickness from 1000 micro at the base to 125 micro at the upper end. This gradient-thickness design allows the separation and densitometric quantitation of very small traces of free fatty acids from relatively large and complex lipid samples in a single chromatographic step. The method has been shown to be applicable directly to the crude total lipid extracts of several mammalian tissues. It appears to generate little if any artifactual free fatty acids from the breakdown of complex lipids, in contrast to the undesirable behavior of silicic acid columns in this respect. Gradient-thickness thin-layer chromatography promises to be useful for the quantitative isolation of trace amounts not only of other types of lipids but also of classes of compounds other than lipids.     

Inactivation of proteins and other biological macromolecules during chromatographic methods of bioseparation.

The denaturation of proteins and other biological macromolecules such as gentamycin, mRNAs, and long-chain fatty acids during their separation by different chromatographic techniques is analyzed. Non-conventional techniques such as centrifugal partition chromatography are also examined. Particular attention is paid to the denaturing mechanisms prevalent under processing conditions, and how denaturation may perhaps be alleviated under laboratory conditions or during scale-up. The available mechanistic studies shed physical insights into the conformational behavior of proteins on chromatographic columns. Mechanistic studies of other biological macromolecule separation on columns is rare. Numbers for both recovery and purity of the biological product are presented wherever available. Scale-up studies are rare, nevertheless, those that are presented together do provide significant and valuable information, and may be generalized to other systems with caution.     

Purification of gangliosides by liquid-liquid partition chromatography

A liquid-liquid partition chromatographic technique was applied to separate amphiphilic glycolipids. A two-phase solvent system composed of n-butanol-t-butyl methyl ether-acetonitrile-water at a volume ratio of 3:1:1:5 was found to be suitable for separating the gangliosides from total lipids extracted from rat brain by liquid-liquid partition chromatographic systems, namely centrifugal partition chromatography (CPC) and high-speed counter-current chromatography. GM1 could be separated rapidly by using the upper phase as stationary phase for both systems. Moreover, various kinds of gangliosides (GM1, GD1a, GD1b, GT1b) could be separated individually by using the lower phase as stationary phase by CPC. The sample can be recovered without loss by these systems.     

Chloroplast and cytoplasmic low-molecular-weight ribonucleic acid components of the leaf of Vicia faba L

1. A method for the extraction of plant nucleic acids and their separation on methylated-serum-albumin-kieselguhr columns is described. It is demonstrated that the characteristics of the elution profiles of material from the same source are consistently reproducible. 2. Major dissimilarities were found in the elution profiles of nucleic acids from root and from leaves of Vicia faba L. These dissimilarities were confirmed by polyacrylamide-gel electrophoresis. 3. Four distinct types of low-molecular-weight RNA were demonstrated to be present in leaves, clearly distinguished by their behaviour when chromatographed on methylated-serum-albumin-kieselguhr columns. (a) Both cytoplasmic and chloroplast ribosomes contained a low-molecular-weight RNA, and these components were distinct from each other. (b) The chloroplast possessed a unique ;soluble' RNA (i.e. RNA that is not precipitated by centrifugal forces that sediment ribosomes) which was not present in the rest of the cell. (c) A soluble component, probably transfer RNA, was found in both the chloroplasts and in the cytoplasm. 4. The components distinguishable by methylated-serum-albumin-kieselguhr column chromatography could not be distinguished by sucrose-density-gradient centrifugation.     

Gel permeation chromatography of neutral hydroxy lipids on Sephadex LH-20

Gel-permeation chromatography on Sephadex LH-20, using ethanol as eluent, permits the resolution of neutral hydroxy lipids according to molecular size. The influence of molecular shape, functional groups, chain lengths, and degree of unsaturation, as well as the effect of the eluent on the elution pattern are discussed. The usefulness of the method for the separation of classes of hydroxy lipids which cannot be resolved by other chromatographic procedures, is demonstrated. Examples include the separations of 1,2- and 1,3-diglycerides from long-chain alcohols, and of alkyl ethanediol monoethers from cholesterol.     

Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5

Highly selective chromatography of microsomal enzymes has been carried out on columns of immobilized cytochrome b5 that was obtained by detergent solubilization (d-b5) of the complete amphipathic molecule. Several partially purified isozymes of cytochrome P-450 are resolved on d-b5 columns, and one high-affinity isozyme has been readily purified to homogeneity. Chromatographic selectivity and correlation of elution order of isozymes of cytochrome P-450 with direct spectral measurements of affinity constants suggests affinity chromatography on d-b5 columns. Substantial one-step enrichments of NADH-cytochrome-b5 reductase and an unstable cytochrome b5-dependent oxidase of cholesterol synthesis, 4-methyl sterol oxidase, have been obtained on d-b5 columns which further supports this conclusion. Comparison of chromatographic behavior on columns of immobilized cytochrome b5 that was obtained by trypsin solubilization (t-b5) with d-b5 columns shows marked differences which must be attributed to the absence of the hydrophobic domain of the t-b5 molecule. NADH-cytochrome-b5 reductase and the high affinity isozyme of cytochrome P-450 purified by d-b5 affinity chromatography are poorly retained on t-b5 columns. A different cytochrome P-450 isozyme with lower affinity for cytochrome b5 is only retained on d-b5 columns. Cytochrome-P-450 reductase is not retained on either column. Because affinity chromatography is suggested on d-b5 columns, the procedure may be generally applicable for predicting protein-protein interactions of microsomal electron transport components that either donate electrons to, or receive electrons from, cytochrome b5. In addition, the procedure should have considerable utilitarian application for enzyme enrichment.     

Two-dimensional chromatography on silica gel-loaded paper for the microanalysis of polar lipids

Two-dimensional chromatography on commercially available silica gel-loaded paper for the microanalysis of polar lipids from various tissues is described. All common phospholipids and their lyso derivatives can be reproducibly separated. As many as 22 lipid components were separated on a single chromatogram. Improved methods for staining lipids and for determining phosphorus in the chromatographic spots are reported.     

The purification and properties of carbonic anhydrases from guinea-pig erythrocytes and the mucosae of the gastrointestinal tract

Procedures for isolating carbonic anhydrase (EC 4.2.1.1) enzymes from the erythrocytes and the mucosae of the gastrointestinal tract of guinea pigs are described. From a haemolysate, haemoglobin was removed by the addition of ammonium sulphate, and also by two other methods, namely by gel filtration or by adsorption on DEAE-Sephadex. The crude enzyme thus obtained was resolved into the different isoenzymes by chromatography with DEAE-cellulose. From particle-free supernatants of homogenates of some gastrointestinal tissues, carbonic anhydrases were purified by ammonium sulphate fractionation, gel filtration, and ion-exchange chromatography with DEAE-cellulose. The major isoenzymes from blood, stomach, proximal colonic mucosa and caecal mucosa were homogeneous during ion-exchange chromatography, acrylamide-gel electrophoresis, and centrifugal examination. From these tissues, carbonic anhydrase was isolated as two major isoenzymes. They resemble the pairs of isoenzymes discovered in the bloods of other species. The carbon dioxide hydratase activity of one isoenzyme (;high activity' carbonic anhydrase) was 40 times that of the other isoenzyme (;low activity' carbonic anhydrase), as measured at a single substrate concentration. Two other minor components of the enzyme are also found in guinea-pig erythrocytes. All of the enzymes isolated had molecular weights of nearly 30000 (sedimentation equilibrium). ;High activity' carbonic anhydrases from blood and gastrointestinal tissues were indistinguishable according to some chemical, physical and kinetic measurements; similarly ;low activity' carbonic anhydrases from those tissues were indistinguishable. ;High activity' carbonic anhydrase was markedly different from the ;low activity' carbonic anhydrase with respect to its amino acid composition, chromatographic behaviour and isoelectric pH value. Marked differences were also found in the tissue concentrations of the major isoenzymes. It is suggested that the characteristic and selective distribution of the different forms of carbonic anhydrase in the guinea-pig tissues is related to the specific and different physiological functions of the enzymes.     

Gas—liquid chromatography of isobutyl ester,N(O)-heptafluorobutyrate derivatives of amino acids on a glass capillary column for quantitative separation in clinical biology

A gas chromatographic method adapted to routine analysis has been developed for quantitative separation on glass capillary columns for free proteic and other known amino acids normally or abnormally found in physiological fluids. The procedure involves ion-exchange chromatography and isobutyl ester, N(O)-heptafluorobutyrate derivatization of free plasma and urine amino acid samples. Derivatized components were ascertained by combined gas chromatography—mass spectrometry. The use of glass for the capillary column is mandatory to achieve qualitative and quantitative analysis of the known occurring amino acids in urine and small plasma samples. Quantitative analysis of several types of human amino acid disorders are presented.     

The Mycobacterium tuberculosis pks2 gene encodes the synthase for the hepta- and octamethyl-branched fatty acids required for sulfolipid synthesis

Multidrug-resistant tuberculosis is a major global health emergency. Cell wall lipids of Mycobacterium tuberculosis can play crucial roles in the pathogenesis. The enzymes involved in their synthesis can be ideal new drug targets against tuberculosis, because many such lipids are unique to this pathogen. A variety of multiple methyl-branched fatty acids are among such unique lipids. We have identified seven genes highly homologous to the mas gene, which is known to be involved in the production of one class of such multiple methyl-branched fatty acids. One of these mas-like genes, pks2, was disrupted using a phage-mediated delivery of the disruption construct. Gene disruption by homologous recombination was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Thin-layer and radio gas-chromatographic analyses of lipids derived from [1-14C]propionic acid and gas chromatography/mass spectrometry analysis of the fatty acids and hydroxy fatty acids showed that the pks2 mutant was incapable of producing hepta- and octamethyl phthioceranic acids and hydroxyphthioceranic acids that are the major acyl constituents of sulfolipids. Consequently, pks2 mutant does not produce sulfolipids. Sulfolipid deficiency in pks2 mutant was confirmed by two-dimensional thin-layer chromatographic analysis of lipids derived from [1-14C]propionic acid and 35SO4(-2). With this sulfolipid-deficient mutant, it should be possible to test for the postulated important roles for sulfolipids in the pathogenesis of M. tuberculosis.     

Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns

In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the micromolar range. The aim of this study was both to describe a chromatographic assay with an increased efficiency of the dinucleoside separation, which enables the reduction of analytical run times, and to establish a chromatographic assay using conditions, which allow MALDI-mass spectrometric analysis of the resulting fractions. We compared the performance of conventional silica reversed phase chromatography columns, a perfusion chromatography column and a monolithic reversed-phase C18 chromatography column. The effects of different ion-pair reagents, flow-rates and gradients on the separation of synthetic diadenosine polyphosphates as well as of diadenosine polyphosphates isolated from human platelets were analysed. Sensitivity and resolution of the monolithic reversed-phase chromatography column were both higher than that of the perfusion chromatography and the conventional reversed phase chromatography columns. Using a monolithic reversed-phase C18 chromatography column, diadenosine polyphosphates were separable baseline not only in the presence of tetrabutylammonium hydrogensulfate (TBA) but also in the presence of triethylammonium acetate (TEAA) as ion-pair reagent. The later reagent is useful because, in contrast to TBA, it is compatible with MALDI mass-spectrometric methods. This makes TEAA particularly suitable for identification of unknown nucleoside polyphosphates. Furthermore, because of the lower backpressure of monolithic reversed-phase chromatography columns, we were able to significantly increase the flow rate, decreasing the amount of time for the analysis close to 50%, especially using TBA as ion-pair reagent. In summary, monolithic reversed phase C18 columns markedly increase the sensitivity and resolution of dinucleoside polyphosphate analysis in a time-efficient manner compared to reversed-phase perfusion chromatography columns or conventional reversed-phase columns. Therefore, further dinucleoside polyphosphate analytic assays should be based on monolithic silica C18 columns instead of perfusion chromatography or conventional silica reversed phase chromatography columns. In conclusion, the use of monolithic silica C18 columns will lead to isolation and quantification of up to now unknown dinucleoside polyphosphates. These chromatography columns may facilitate further research on the biological roles of dinucleoside polyphosphates.     

High-pressure liquid chromatographic analysis of amino acid phenylthiohydantoins: Comparison with other techniques

High-pressure liquid chromatography, utilizing reverse phase μ Bondapak C₁₈ columns and elution with increasing acetonitrile concentrations, has been used to resolve amino acid phenylthiohydantoins obtained from the automated Edman degradation of proteins. Assignment of identity to residues which are difficult to distinguish or identify conclusively by other conventional techniques is easily achieved by high-pressure liquid chromatographic techniques. The use of high-pressure liquid chromatography, in parallel with gas-liquid and polyamide thin-layer chromatography, allows unequivocal assignments of identity to amino acid phenylthiohydantoins obtained in protein sequencing. Single protein sequence determinations can be extended by 20 to 100% by the use of high-pressure liquid chromatography with rapid, accurate, and quantitative identifications of amino acid phenylthiohydantoins.     

Fractionation of plant aminoacyl-tRNA synthetases on tRNA-Sepharose columns.

It has been shown that tRNA-Sepharose, a chromatographic adsorbent containing unfractionated tRNA bound to a Sepharose matrix, is a useful, group-specific adsorbent for fractionation of the plant aminoacyl-tRNA synthetases. Conditions are described in which Val-, Trp-, Phe-, Leu- and Ile-tRNA synthetases from yellow lupin seeds can be separated from each other on the tRNA-Sepharose columns. Factors affecting affinity chromatography on the t-RNA-Sepharose columns are discussed. The affinity chromatography procedure for the purification of lupin Ser-tRNA synthetase to homogenity is described.     

High-throughput screening of packed-bed chromatography coupled with SELDI-TOF MS analysis: monoclonal antibodies versus host cell protein

A feasibility study to couple high throughput screening of packed bed chromatography with mass spectrometric detection by SELDI-TOF MS is presented. As model system monoclonal antibodies (mAb) versus host cell protein (HCP) from an industrial cultivation was chosen. Packed bed chromatography was screened on a TECAN Evo Freedom 200 station using miniaturized chromatographic columns placed on a specially designed array carrier linked to a commercially available T-Stack module. Gradient elution of the bound proteins was performed by applying a multiple step strategy. When analyzing selected HCP peaks as well as the detected antibody peaks throughout the chromatographic runs a direct correlation between applied and detected components was established. The sensitivity of conventional protein A chromatography was found to be lower than SELDI-TOF MS analysis. During initial screening a shift in the elution pattern for one of the monoclonal antibodies detected with all four resins was identified to be a heterogeneity in the mAb glycosylation pattern. In addition, a detailed differentiation between various HCP fractions through out the chromatographic process using SELDI-TOF analysis let to the detection of HCP components possibly adhering to the mAbs during chromatographic separations.     

Chromatographic and electrophoretic approaches in ink analysis

Inks are manufactured from a wide variety of substances that exhibit very different chemical behaviors. Inks designed for use in different writing instruments or printing methods have quite dissimilar components. Since the 1950s chromatographic and electrophoretic methods have played important roles in the analysis of inks, where compositional information may have bearing on the investigation of counterfeiting, fraud, forgery, and other crimes. Techniques such as paper chromatography and electrophoresis, thin-layer chromatography, high-performance liquid chromatography, gas chromatography, gel electrophoresis, and the relatively new technique of capillary electrophoresis have all been explored as possible avenues for the separation of components of inks. This paper reviews the components of different types of inks and applications of the above separation methods are reviewed.     

Lipids of Leaves and Seeds

The lipids in rice bran lipoprotein were separated and their qualitative and quantitative analyses were carried out by column, thin-layer, gas-liquid, and paper chromatographic methods. More than twenty lipid components were detected, of which triglycerides and glycolipids were found to be the major components. Considerable amounts of free fatty acids were also found. The glycolipids were composed of mono- and digalactosyl glycerides, sterol glycosides and their esters, probably phytocerebrosides, and other unknown glycolipids. Phospholipids were present as the minor components of rice bran lipoprotein. The presence of oryzanols in the lipoprotein was observed.     

Characterization of enantioselective binding of racemic natural tetrahydropalmatine to DNA by chromatographic methods

A racemate from natural product, tetrahydropalmatine (THP), was characterized on its enantioselective binding to DNA by the chromatographic methods including microdialysis/HPLC, centrifugal ultrifiltration/HPLC and immobilized DNA affinity chromatography. It was found that its (+)-enantiomer was preferential to binding on B-form duplex DNA including calf thymus DNA, AT and GC sequence oligo DNA, as well as triplex oligo DNA. The binding constants of the THP enantiomers to ct-DNA were determined with the methods of microdialysis/HPLC and frontal affinity chromatography. In addition, the DNA structural preference of either enantiomer was evaluated with the chromatographic methods.     

The stepwise removal of histones from chicken erythrocyte nucleoprotein

1. A fractionation of chicken erythrocyte histones was achieved simultaneously with their extraction from saline-washed nuclei by stepwise titrations to progressively lower pH values. 2. Different acids and dilute buffer solutions of comparable pH behaved similarly in stepwise extractions of histones. 3. The histone preparations so obtained were characterized by their amino acid composition and behaviour on zone electrophoresis in starch gels. 4. The fractionation by titration was quite sharp at appropriate pH ranges, and the histone fraction that is apparently unique to avian erythrocytes was obtained without contamination by other histone fractions. 5. Histones prepared by stepwise titration were fractionated further by cation-exchange and exclusion chromatography. The chromatographic behaviour and amino acid composition of the components permitted comparison with histones prepared by other methods. 6. Histone fraction IIb was resolved into its subfractions IIb(1) and IIb(2) by exclusion chromatography on Bio-Gel P-60. 7. Histone fractions III and IV, previously reported to be absent from chicken erythrocyte nuclei, were found in extracts made at pH1.     

Survey of recent advances in analytical applications of immunoaffinity chromatography

Methods that use immunoaffinity chromatography (IAC) for sample preparation or detection are becoming increasingly popular as tools in the analysis of biological and nonbiological compounds. This paper presents an overview of immunoaffinity chromatography and examines some recent developments of this technique in analytical applications. The emphasis is placed on HPLC-based IAC methods or those that combine IAC with other instrumental techniques; however, novel approaches that employ low-performance IAC columns for chemical quantitation are also considered. Particular applications that are examined include (1) the use of IAC in the direct detection of analytes, (2) the extraction of samples by IAC prior to on- or off-line detection by other methods, (3) the use of IAC in chromatographic-based immunoassays, and (4) the development of postcolumn reactors based on IAC for the detection of analytes as they elute from other types of chromatographic columns. The advantages and limitations for each approach are considered. In addition, a summary is provided of reports in the literature that have used IAC for these various formats.