Resolution of allelic and non-allelic variants of histone H1 by cation-exchange-hydrophilic-interaction chromatography.

A mixed-mode high-performance liquid chromatography (HPLC) method that resolves the six known non-allelic variants of chicken erythrocyte histone H1 is described. Common, but previously unknown, allelic variants of H1 that comigrate in polyacrylamide gel electrophoresis are also resolved. The resolution of H1 variants achieved by this method should be useful in determining the functional significance of H1 sequence heterogeneity and in analyses of post-translational modification of H1. Furthermore, the principles behind the separation should be applicable to analyses of polymorphism in other proteins.

Hydrophilic-interaction chromatography of complex carbohydrates.

Complex carbohydrates can frequently be separated using hydrophilic-interaction chromatography (HILIC). The mechanism was investigated using small oligosaccharides and a new column, PolyGLYCOPLEX. Some carbohydrates exhibited anomer separation, which made it possible to determine the orientation of the reducing end relative to the stationary phase. Amide sugars were consistently good contact regions. Relative to amide sugars, sialic acids and neutral hexoses were better contact regions at lower levels of organic solvents than at higher levels. HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using volatile mobile phases. This was evaluated with native glycans and with glycans derivatized with 2-aminopyridine or a nitrobenzene derivative. Both asialo- and sialylated glycans could be resolved using the same set of conditions. With derivatized carbohydrates, detection was possible at the picomole level by UV detection or on-line electrospray mass spectrometry. Selectivity compared favorably with that of other modes of HPLC. HILIC is promising for a variety of analytical and preparative applications.

Use of hydrophilic interaction chromatography for the study of tyrosine protein kinase specificity.

A new HPLC method has been developed to assay tyrosine protein kinase activity. Using hydrophilic interaction chromatography, it is possible to resolve the four components of the incubation medium: substrate peptide, [32P]phosphorylated peptide, unreacted [gamma-32P]ATP, and 32P-labelled inorganic phosphate. ATP interacts so strongly with the stationary phase material that it can be removed selectively from the incubation medium with solid-phase extraction cartridges packed with the same type of material. The three remaining components of interest can then be resolved by reversed-phase or hydrophilic interaction HPLC. This procedure permits the evaluation of almost every type of peptide as a substrate of tyrosine protein kinase.

Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds.

When a hydrophilic chromatography column is eluted with a hydrophobic (mostly organic) mobile phase, retention increases with hydrophilicity of solutes. The term hydrophilic-interaction chromatography is proposed for this variant of normal-phase chromatography. This mode of chromatography is of general utility. Mixtures of proteins, peptides, amino acids, oligonucleotides, and carbohydrates are all resolved, with selectivity complementary to those of other modes. Typically, the order of elution is the opposite of that obtained with reversed-phase chromatography. A hydrophilic, neutral packing was developed for use in high-performance hydrophilic-interaction chromatography. Hydrophilic-interaction chromatography is particularly promising for such troublesome solutes as histones, membrane proteins, and phosphorylated amino acids and peptides. Hydrophilic-interaction chromatography fractionations resemble those obtained through partitioning mechanisms. The chromatography of DNA, in particular, resembles the partitioning observed with aqueous two-phase systems based on polyethylene glycol and dextran solutions.

Hydrophobic interaction chromatography of peptides as an alternative to reversed-phase chromatography.

Hydrophobic interaction chromatography (HIC) was examined as an alternative to reversed-phase chromatography (RPC) for peptide separations by high-performance liquid chromatography. With small peptides, selectivity was similar in both modes. This was the case with commercially available standards and with a set of synthetic peptides having the same amino acid composition but different sequences. Column efficiency was higher in RPC. HIC possesses several other disadvantages, including significant baseline changes during gradient elution and a requirement for non-volatile mobile phases, which complicates peptide isolation. Thus, RPC is still the method of choice for most small peptides. Marked differences in selectivity were noted with small proteins and polypeptides large enough to possess tertiary structure. Good results were also obtained by HIC in the case of some peptides that could not be purified at all by RPC, due to aggregation or poor binding or recovery. Thus, in these cases, HIC is a useful alternative to RPC for peptide purification.

Cation-exchange chromatography of peptides on poly(2-sulfoethyl aspartamide)-silica.

A strong cation-exchange material, poly(2-sulfoethyl aspartamide)-silica (PolySULFOETHYL Aspartamide) was developed for purification and analysis of peptides by high-performance liquid chromatography. All peptides examined were retained at pH 3, even when the amino terminus was the only basic group. Peptides were eluted in order of increasing number of basic residues with a salt gradient. Capacity was high, as was selectivity and column efficiency. This new column material displays modest mixed-mode effects, allowing the resolution of peptides having identical charges at a given pH. The selectivity can be manipulated by the addition of organic solvent to the mobile phases; this increases the retention of some peptides and decreases the retention of others. The retention in any given case may reflect a combination of steric factors and non-electrostatic interactions. Selectivity was complementary to that of reversed-phase chromatography (RPC) materials. Excellent purifications were obtained by sequential use of PolySULFOETHYL Aspartamide and RPC columns for purification of peptides from crude tissue extracts. The new cation exchanger is quite promising as a supplement to RPC for general peptide chromatography.

Detection of oxidized and reduced glutathione with a recycling postcolumn reaction.

A rapid, sensitive, and selective method for the quantitation of both oxidized (GSSG) and reduced (GSH) glutathione in biological materials is described. Oxidized and reduced glutathione are resolved by anion-exchange high-performance liquid chromatography and detected with an in-line, recycling postcolumn reaction. The recycling reaction specifically amplifies the response to oxidized and reduced glutathione 20-100 times over that obtained with a stoichiometric reaction, permitting the detection of 2 pmol glutathione. Oxidized and reduced glutathione levels were measured in rat liver and in dog heart mitochondria. Special precautions are necessary to avoid artifacts which lead to either underestimation or overestimation of GSSG levels. GSH/GSSG ratios of approximately 100-300 were observed in samples prepared from rapidly frozen rat liver. Somewhat higher GSH/GSSG ratios were observed in isolated dog heart mitochondria.

High-performance liquid chromatography of human hemoglobins on a new cation exchanger.

We have investigated the use of a high-performance liquid chromatographic (HPLC) column packed with a unique weak cation exchanger prepared by coating silica with poly(aspartic acid) for hemoglobin analysis. The complete separation of hemoglobin Bart's F, A0, A2, S, C, D, E, G, SG, Winnipeg and Sealy was achieved by gradient elution within 30 min. The high resolution made it possible to distinguish hemoglobin variants such as Bart's, AC, AD, AE, AG, AS, ASG, CC, SC, SS, Winnipeg, Sealy and beta-chain variants with thalassemia such as S/beta +, S/beta 0 and S(C)-beta + thalassemia. Comparison of DEAE-cellulose column chromatography and our HPLC method for the quantitation of hemoglobin A2 yielded a good correlation. Hemoglobins A2, C and E are completely resolved on PolyCAT A columns in contrast to both cellulose acetate electrophoresis and DEAE-cellulose column chromatography. The high resolution of the system and the accuracy of the method combined with complete automation make this procedure useful for diagnosis of hemoglobin disorders in both a research and clinical laboratory environment.

Cation-exchange high-performance liquid chromatography of proteins on poly(aspartic acid)-silica.

A simple cation-exchange material for high-performance liquid chromatography of proteins was developed. Poly(succinimide) reacted rapidly with aminopropyl-silica and the product was hydrolyzed to poly(aspartic acid)-silica. Reaction conditions were optimized to yield a material with an ion-exchange capacity of 430 mg hemoglobin/g material. High-performance liquid chromatographic columns of the material featured excellent performance in terms of capacity, selectivity, recovery of enzyme activity, peak shape and durability. Protein standards and clinical hemoglobin samples were well resolved in minutes. Poly(succinimide)-silica was readily derivatized to give products other than poly(aspartic acid)-silica, and several such materials were prepared. Such materials could be useful for affinity chromatography or enzyme immobilization.

Lactate dehydrogenase activity in mouse lung following 1,1-dichloroethylene: index of airway injury.

Lactate dehydrogenase (LD) activity and its isozyme profile in mouse lung homogenate was affected by oral administration of 1,1-dichloroethylene (1,1-DCE). Following 100 mg 1,1-DCE/kg, LD-3 increased significantly. After 200 mg 1,1-DCE/kg, LD-5 increased whereas LD-1 and LD-2 decreased, with a resultant higher M:H ratio than controls. In contrast, elevated LD activity in serum following 1,1-DCE was predominantly associated with striking increases in total activity and changes in isozyme patterns resulting in a decrease in the M:H ratio. LD activity in liver and erythrocytes were unaffected by 1,1,-DCE administration. Although total activity in kidney was decreased, no changes were detected in the isozyme profile. Pulmonary damage induced by 1,1-DCE was reflected in significant increases in total activity and all isozymes in bronchopulmonary lavage fluids. Thus, detection of lung-derived LD activity in lung lavage fluids can be a useful index of pulmonary airway injury.

Apolipoprotein C-III-1 activates lysosomal sphingomyelinase in vitro.

Apolipoprotein (apo)C-III-1 from human very low density lipoprotein stimulates 14-fold the activity of lysosomal sphingomyelinase from human fibroblasts. At the sphingomyelin concentrations tested, maximal stimulation was obtained with 5 microM apoC-III-1 or apoC mixture. Apolipoproteins A-I, A-II, B, and C-I conferred little or no stimulation. Sphingomyelinase was stimulated 20-fold by lysophosphatidylcholine with an optimum concentration of 70 microM using 0.3 mM substrate. Sphingomyelinase activity was inhibited by concentrations of apoC-III-1 and lysophosphatidylcholine three- to fivefold above stimulatory levels. Triton X-100 activated sphingomyelinase 300-fold with a pH optimum of 5.0, while the pH optimum with the biological activators was 4.0. These results raise the possibility of an in vivo activity for the biological activators. The proteins that enter lysosomes as part of a lipoprotein complex may activate lysosomal enzymes that degrade the lipid components.

Rapid assessment of isoenzymes by high-performance liquid chromatography.

We describe the rapid profiling of isoenzymes by use of microparticulate anion-exchange chromatography supports and a continuous, post-separation enzyme detector in a high-performance liquid chromatograph. Chromatographic analysis and enzyme detection are fully automated and provide excellent reproducibility. Factors affecting the isoenzyme profile and detector response characteristics are assessed. Lactate dehydrogenase and creatine kinase isoenzymes in tissue extracts, control materials used as electrophoretic standards, and serum were profiled by this method to establish the resolution and reliability of the method. We show the clinical use of this method in detecting changes in these isoenzymes in serum associated with acute myocardial infarction.