High-performance cation-exchange chromatography of proteins

Approximately one-third of all proteins reported in the literature have a pI sufficiently high to be resolved by cation-exchange chromatography. This paper reports the preparation and use of new high-performance polymeric-bonded-phase cation-exchange columns. Starting from a very stable, covalently bonded polyamide coating on microparticulate silica, simple derivatization produces a versatile cation-exchange material useful for separations traditionally performed on classical carboxymethylated soft gel supports. Column behavior was monitored using chymotrypsinogen, cytochrome c, and lysozyme as standards. The polymeric bonded phase was stable to pH 2.5 and exhibits enhanced selectivity for proteins due to a slight hydrophobic character of the matrix. Several separations of biological interest that demonstrate the utility of these small cation-exchange columns for modern biochemical separations are shown.     

Variables in the high-performance anion-exchange chromatography of proteins

The effect of mobile phase velocity, separation time, support pore diameter, column length, and temperature on resolution and loading capacity of a new commercially available high-performance anion-exchange support, SynChropak AX-300, has been examined. This material is a macroporous spherical silica of 10 μm particle size with a bonded polymeric amine layer. It was found that the heterogeneity of ovalbumin samples, combined with bovine serum albumin, make them useful probes in evaluation of anion-exchange supports. In the columns of 4.1 mm i.d., the highest resolutions of proteins were achieved at a flow rate of 0.25 ml/min. Up to 10 mg of protein per injection could be applied on a 4.1 × 250 mm AX-300 column with good resolution. Columns of 50 mm length had one-tenth the protein load capacity of a 250-mm column, retaining approximately 75% of the resolution.     

A rapid separation of the four major deoxynucleosides and deoxyinosine by high-pressure liquid cation-exchange chromatography

A simple, rapid, and sensitive method for the measurement of DNA, RNA, and protein synthesis in cell samples is presented. The method measures the amount of isotope incorporated into these macromolecules after cell collection on filter paper and washing with a methanol:chloroform:water mixture. Samples as low as 50 μg in size can be prepared in less than 30 sec for measurement of radioactivity.     

A highly sensitive method for the estimation of pyrimidine dimers in DNA by high-pressure liquid cation-exchange chromatography

We have developed a rapid, highly sensitive method for the separation of thymine-dimers from thymine on a cation-exchange resin, which is capable of detecting one dimer out of 5 × 10³ to 10⁴ thymine molecules (depending on the specific activity of the used material) by the means of high-pressure liquid chromatography. The assay has been very useful in DNA-hybridization studies using $\text{(}\text{TT}\text{)}$-containing DNA-strands and there is evidence that the method will be valuable for photoreactivation studies of uv-damaged DNA and for the enzymes involved in dimer-excision studies.     

Separation of icosenoic acids,monohydroxyicosenoic acids,and prostaglandins by high-pressure liquid chromatography on a silver ion-loaded cation-exchange column

Prostaglandins and monohydroxy fatty acids derived from 8,11,14-icosatrienoic acid and arachidonic acid have been separated by high-pressure liquid chromatography using a cation-exchange column loaded with silver ions. The retention times in a variety of solvent systems have been determined for prostaglandin E₁(PGE₁), PGF_1α, PGD₂, PGE₂, PGF_2α, 6-oxoPGF_1α, 15-hydroxy-8,11,13-icosatrienoic acid, 5-hydroxy-6,8,11,14-icosatetraenoic acid, 8-hydroxy-5,9,11,14,-icosatetraenoic acid, 9-hydroxy-5,7,11,14-icosatetraenoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid, 12-hydroxy-5,8,10,14-icosatetraenoic acid, 15-hydroxy-5,8,11,13-icosatetraenoic acid, 8,11,14,-icosatrienoic acid, and arachidonic acid. The mechanisms involved in the interaction of solutes with the stationary phase have been investigated. Retention times on silver ion columns appear to be determined by a combination of interactions between (a) the silver ions of the stationary phase and double bonds of the solute and (b) polar groups of the stationary phase and polar groups of the solute. The relative contributions of these two types of interactions to the retention of solutes can be varied over a wide range by altering the composition of the solvent. In this way the selectivity of the stationary phase can be controlled in order to optimize the separation of any given group of solutes. The maximum separation of solutes on the basis of the number of double bonds they possess is obtained by using polar solvents containing low concentrations of acetonitrile. As the polarity of the mobile phase is reduced or the concentration of acetonitrile increased, the selectivity of the stationary phase tends to resemble that of normal-phase chromatography on silicic acid.     

Separation of phenylthiohydantoin-amino acids by high-pressure liquid chromatography for sequence determination of radiolabeled proteins

Published procedures for the separation of ethyl acetate-extractable phenylthiohydantoin-amino acids by high-pressure liquid ehromatography with organic solvent gradients have been modified to facilitate sequence determination of proteins labeled with several radioactive amino acids at a time.     

High-performance liquid chromatography of peptides on a macroreticular cation-exchange resin: Application to peptide mapping of Bence-Jones proteins

A high-performance liquid chromatography system has been developed for analytical peptide mapping and preparative peptide separation. The method uses a macroreticular cation-exchange resin of the styrene-divinylbenzene type, having a relatively small particle size (6 ± 1 μm) and a high crosslinkage (35%). Elution was performed with a linear gradient of increasing salt and organic solvent concentration for monitoring the effluent at 210 nm. The system showed remarkable peak resolution and excellent mechanical and chemical stability, and high precision analysis of tryptic digests of S-aminoethyl Bence-Jones proteins was achleved in less than 120 min with nanomolar to micromolar quantities of the samples. The isolated peptides could be subjected to subsequent chemical determinations directly or after simple desslting process.     

Separation of hemoglobin types by cation-exchange high-performance liquid chromatography

The use of a recently developed cation-exchange HPLC packing material for the separation of hemoglobin types in human blood has been investigated. Adult and newborn hemolysates from normal individuals and from subjects with hemoglobin disorders were analyzed using a weak cation carboxymethyl-bonded phase on 5-micron-particle-size silica. Elution was accomplished using a Bistris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1, 3-propanediol) gradient. Seven well-resolved HbA1 fractions eluted before the major HbA peak. Hbs A1a, A1b, A1c and an HbA1 fraction that increased with aging of the hemolysates were separately eluted. HbF when present or when added to the hemolysates eluted as a distinct peak. HbA was followed by Hbs A2, S, and C when present. An early-eluting peak corresponding to Hb Bart's was identified in newborn hemolysates. It is concluded that cation-exchange HPLC provides a new tool for the reliable separation of minor hemoglobin components.     

Molecular weight separation of proteins and peptides with a new high-pressure liquid chromatography column.

A new high-pressure liquid molecular weight chromatography column was evaluated for its ability to separate proteins and peptides. The column was able to give a linear separation of compounds between 5,000–700,000 M_r. Chromatography of posterior pituitary extracts, tumor-associated fetal antigens, and estrogen receptors demonstrated the ability of the column to separate biological samples.     

Separation of lipid-free egg yolk proteins by high-pressure liquid chromatography using solvents containing formic acid

A method for obtaining total protein patterns from lipid-containing systems, in particular egg yolk, is described. After dispersion of the yolk in 8 M guanidine hydrochloride solution, lipid is removed by extraction with chloroform-methanol and petrol. The protein solution is applied to a high-pressure liquid chromatograph and eluted with a gradient of formic acid, isopropanol, and acetonitrile. In measurements on a known yolk protein, duck apovitellenin I, the method did not cause irreversible formylation of N-terminal or other residues. The method was used (1) to compare protein patterns of whole yolk from hen and quail eggs; (2) to isolate and partially sequence quail apovitellenin I; and (3) to compare protein patterns of "white yolk" and "yellow yolk."     

Determination of glutathionyl-hemoglobin in human erythrocytes by cation-exchange high-performance liquid chromatography

Since glutathionyl-hemoglobin has been suggested to be a clinical marker of oxidative stress in human blood and given the growing biological relevance of oxidative stress as a pathogenic factor in several diseases, we describe a method to measure glutathionyl-hemoglobin concentration in erythrocytes, by using cation-exchange high-pressure liquid chromatography with UV detection. The glutathionyl-hemoglobin peak has been identified on the basis of the following findings: (a) the peak increased when the sample was incubated with oxidized glutathione; (b) the peak disappeared when the sample was reduced with dithiothreitol, with the simultaneous increase of that corresponding to hemoglobin A(0); (c) the peak could be detected by incubating hemoglobin A(0) with reduced glutathione; (e) deconvoluted mass spectrum of the glutathionyl-hemoglobin peak showed a 16172.0-Da molecular mass, corresponding to hemoglobin beta bound to glutathione. Glutathionyl-hemoglobin concentration has been determined in erythrocytes of 40 healthy subjects, with a mean value of 2.58+/-0.7%, calculated as the percentage of its peak area ratio to that of total hemoglobin (HbA(0)+HbA(2)+HbA(1C)+glutathionyl-hemoglobin). The availability of a simple and reproducible method to detect glutathionyl-hemoglobin concentration in blood could be useful in monitoring oxidative stress, and for investigating the efficacy of antioxidant therapies in clinical trials.     

Protected amino acids as novel low-molecular-weight displacers in cation-exchange displacement chromatography

Although the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of pharmaceuticals, a major impediment to the implementation of displacement chromatography has been the lack of suitable displacer compounds. An important recent advance in the state of the art of displacement chromatography has been the discovery that low-molecular-weight dendritic polymers can be successfully employed as displacers for protein purification in ion-exchange systems. In this article, protected amino acid esters (based on arginine and lysine) are shown to be useful displacers for protein purification in cation-exchange systems. A dynamic affinity plot is employed to evaluate the affinity of these low-molecular-weight compounds under dis-placement conditions. In contrast to large polyelectroyte displacers, the efficacy of these low-molecular-weight displacers was shown to be dependent on both the initial carrier salt concentration and the displacer concentration. In addition to the funcamental interest generated by low-molecular-weight displacers, it is likely that these displacers will have significant operatioal advantages as compared with large polyelectrolyte displacers. (c) 1995 John Wiley & Sons, Inc.     

Quantitation of oxypurines and allopurinol metabolites in biological fluids by cation-exchange chromatography

Chromatography of urine, cerebrospinal fluid, and deproteinized serum on 30 × 0.9 cm columns of AG 50 permits the convenient separation of hypoxanthine, xanthine, allopurinol, and its metabolites. Quantitation by determination of absorbance at 250 mμ is accurate and can be automated. Column preparation is simple, and the columns can be regenerated and used daily for several months with no loss of resolution.     

Analysis of leukotrienes by high-pressure liquid chromatography

A method is described for the partial synthesis of saturated mixed-chain phosphatidylcholines of a high degree (typically 99 mol%) of purity. This procedure has been designed to eliminate the contamination of the mixed-chain product by symmetric chain phosphatidylcholine and the mixed-chain isomer of the desired product, the two principal impurities introduced by previous techniques. This high degree of purity is obtained by employing a method designed for the complete enzymatic hydrolysis of the C-2 fatty acyl moiety in saturated symmetric phosphatidylcholines and a new technique for the acylation of lysophosphatidylcholines employing the catalyst 4-pyrrolidinopyridine. The versatility of this new procedure is illustrated with the synthesis of several saturated mixed-chain phosphatidylcholines.     

Separation of the natural retinoids by high-pressure liquid chromatography

A reverse phase high-pressure liquid chromatography system for rapid separation of various retinoids (vitamin A and its analogs) with little or no degradation is described. This method permits detection of as little as 22 pmol of retinoic acid. The procedure has been applied to the study of retinoic acid metabolism in vitamin A-deficient hamsters.