Preparative high-performance liquid partition chromatography of proteins.

Methods for preparative high-performance liquid chromatography (hplc) of proteins are described. Both normal and reverse-phase chromatography were studied and adapted to the fractionation of proteins in quantities of up to 50 mg. Lichrosorb Diol was used as a “normal phase” for chromatography of hydrophobic proteins. Lichrosorb RP-8 was used for reversephase chromatography of proteins.     

Preparative high-performance liquid chromatographic separation of proteins with HyperD ion-exchange supports

HyperD ion-exchange media combine the mechanical strength of a rigid polystyrene-mineral composite skeleton with the high protein-binding capacity of a three-dimensional soft gel located inside the skeleton. The skeleton solid matrix is completely filled with functionalized, highly hydrophilic, chemically stable ion-exchange hydrogels. These materials gave very efficient columns for protein separation with superior dynamic capacity, high resolving power and excellent protein recovery. Various protein mixtures were used to study the chromatographic performance of these new stationary phases. Comparisons between different particle size packing materials demonstrated the potential of this ion-exchange material for use on a large scale.     

Ionic-exchange high-performance liquid chromatography of Escherichia coli ribosomal small-subunit proteins

Ion-exchange high-performance liquid chromatography was applied to the separation of proteins from the 30S ribosomal subunit. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis analysis. The purification has been made using either unmodified proteins or proteins specifically labeled at their SH group. The results clearly show that the method can be used to purify and identify ribosomal proteins.     

Reverse-phase high-performance liquid chromatography of Escherichia coli ribosomal small subunit proteins

Reverse-phase high-performance liquid chromatography has been explored as an approach for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes. The majority of these proteins are of similar molecular weight and isoelectric point, making separation by size exclusion or ion exchange difficult. With the use of an octadecasilyl silica column and a trifluoroacetic acid-acetonitrile solvent system, the 21 proteins of the 30 S subunit have been separated into 15 peaks. The yield of total protein recovered from the column was ≥85%. The proteins present in each peak have been identified by polyacrylamide gel electrophoretic analysis of the peaks as well as by comparison with the relative retention volumes of known purified 30 S proteins on the column. The results clearly show that this method is a powerful and rapid technique for the identification and purification of 30 S proteins. Analysis of [³H]puromycin-labeled 30 S subunit protein provides an illustrative example of its utility for affinity labeling studies.     

Preparative high-performance liquid chromatography of minor products ofStreptomyces cinnamonensis

Analytical and preparative high-performance liquid chromatography of 3 phenazines and furonaphthoquinone derivative on resersed-phase column are described. The mobile phase was methanol and water. The injected amount of the mixture was about 30 mg for a preparative chromatographic run requiring 80 min. Substances were detected directly in the column effluent by UV detection.     

Purification of synthetic oligodeoxyribonucleotides by ion-exchange high-performance liquid chromatography

Synthetic oligodeoxyribonucleotides rangin from 11 to 37 nucleotides in length and with varying base compositions, prepared by both the phosphotriester and phosphite procedures, have been purified by ion-exchange high-performance liquid chromatography on Whatman Partisil 10/SAX columns using phosphate buffer gradients. The effects of different buffer systems on elution times and resolution have been evaluated. Oligomer composition and length had a marked effect on the resolution achieved. In general the use of formamide buffers gave the best results, particularly in the case of 2′-deoxyguanosine-rich sequences. These methods have also been successfully applied to the purification of mixtures of synthetic oligodeoxynucleotides.     

Determination of proton dissociation constants by ion-exchange high-performance liquid chromatography

The common methods to determine dissociation constants of solutes, e.g., uv spectrophotometry, potentiometry, and conductimetry, are accurate but require at least 1 nmol of compound. High-performance liquid chromatography (HPLC) allows 1 pmol of a uv-absorbing compound to be detected. By adjusting the polarity of the mobile phase, reverse and normalphase properties of an ion-exchanger can be minimized, resulting in a high correlation between charge and retardation of the solute. Thus, the degree of ionization of several compounds was monitored in mobile-phase compositions of different pH values using cation exchange. The pK values of several pterin derivatives corresponded to those obtained by other methods. In addition, pK values of two unidentified pterin derivatives were determined, using only 20 pmol of each.     

Purification of reduced nicotinamide adenine dinucleotide by ion-exchange and high-performance liquid chromatography

Combining ion-exchange (AG MP-1) and reversed-phase (C-18) partition chromatography accomplishes a higher degree of purification of NADH than either method can provide alone. Final elution in 95% ethanol, dehydration with anhydrous sodium sulfate, and storage in dry 1,2-propanediol over molecular sieves prevents hydrolysis of the purified dinucleotide.     

Characterization of uterine estrogen receptors by size-exclusion and ion-exchange high-performance liquid chromatography

This report presents the first application of ion-exchange high-performance liquid chromatography in the study of ER from the rabbit uterus. In the presence of sodium molybdate (20 mM), native ER was eluted as a sharp peak at 0.29 M NaCl by a linear salt gradient, but without molybdate, it resolved into 4 major peaks. Molybdate-stabilized ER from the DEAE column, similar to ER from crude cytosol, sedimented at the 6-8S region in low salt and 4S region in high salt linear sucrose gradients, and was excluded from size-exclusion HPLC. In contrast, dissociated ER subunits from DEAE eluates ranged from 3.5 to 4.5S, and showed differences in molecular weights in a size-exclusion column. These results show that the native ER is a large molecule which dissociates into smaller subunits in the absence of molybdate; each of the steroid-bound moieties differs in molecular weight and surface charge from the native molecule.     

Use of a volatile buffer system in ion-exchange high-performance liquid chromatography of oligonucleotides

A volatile buffer has been adapted for use with ion-exchange high-performance liquid chromatography in the analysis and preparation of oligonucleotides. The system employs a commercial weakly basic anion-exchange column containing a DEAE-derivatized silica gel and eluted with a volatile buffer gradient of triethylamine acetate and acetonitrile. Nucleic acid digests and oligonucleotides synthesized by chemical or enzymatic methods can be analyzed or purified with nearly quantitative recovery following solvent volatilization.     

Supports for reverse-phase high-performance liquid chromatography of large proteins

Reverse-phase high-performance liquid chromatography supports have been developed for use in separating proteins up to 300,000 M_r. They are based on silica supports to which octyl, cyanopropyl, or diphenyl groups are covalently bonded. Their effectiveness in rapidly separating several standard proteins is demonstrated. Applications presented include the separation of the α₁ and α₂ chains of chick Type I collagen within 1 h and the separation of the α and β components of human Type I collagen.     

Hydroxyapatite high-performance liquid chromatography: column performance for proteins

Hydroxyapatite columns for high-performance liquid chromatography that are reusable for a long time were developed; performance tests were carried out by using several types of protein. Using a high flow rate, a sharp chromatographic peak can be obtained for a homogeneous molecule. A very high level of chromatographic separation can be achieved by decreasing the slope of the gradient and increasing the total column length at the same time.     

Separation of tryptic phosphopeptides of ribosomal origin by reversed-phase high-performance liquid chromatography

Phosphorylation sites for cyclic AMP-dependent kinase in ribosomal proteins and their synthetic analogues were converted to tryptic phosphopeptides and analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) using gradients of acetonitrile in water and 0.1% trifluoroacetic acid. Tryptic variants differing by only NH₂-terminal basic amino acid redidues or phosphoryl groups were not always well resolved under these conditions. The different phospho forms could be resolved by RP-HPLC and thin-layer cellulose mapping was found to be the most effective strategy for the absolute purification of trytic phosphopeptides from crude tryptic digests.     

Preparative high-performance liquid chromatography purification of polyunsaturated phospholipids and characterization using ultraviolet derivative spectroscopy

A preparative reversed-phase HPLC system utilizing an isocratic mobile phase to purify up to 10-mg quantities of phospholipids is described. The method was developed to separate oxidation products of polyunsaturated phospholipids from intact, parent lipids. The method is useful for phosphatidylcholine and phosphatidylethanolamine on a preparative scale and for phosphatidylserine and phosphatidic acid on an analytical scale. Both intact phospholipids and oxidized phospholipids were monitored by absorbance at 206 nm. The oxidation products were simultaneously monitored at 234 nm where the intact phospholipids have only a very slight end absorption. Second-derivative uv spectroscopy proved to be extremely useful to identify the presence or to verify the absence of oxidation products in phospholipid samples. For autoxidized docosahexaenoic acid containing phospholipids, the absorbance maximum of diene oxidation products is 237 nm for the trans,trans (t,t) isomer and 246 nm for the cis,trans (c,t) isomers. Similarly, five classes of triene oxidation product stereoisomers have distinct absorbance maxima detected by second-derivative spectroscopy ranging from 269 to 292 nm.     

Preparative steps necessary for the accurate measurement of malondialdehyde by high-performance liquid chromatography.

The need for a more specific, reliable, and reproducible technique for the measurement of malondialdehyde (MDA) has prompted modifications of currently available methods based on the formation and recovery of the complex between MDA and thiobarbituric acid (TBA). To 500 microliters of plasma or to 300 mg of liver homogenate, 2 ml of H2O and 500 microliters of 0.5% butylated hydroxytoluene in methanol were added to prevent further formation of MDA. Precipitation of proteins carried out with 200 microliters of 0.66 N H2SO4 and 150 microliters of 10% Na2WO4 (w/v) led to complete recovery of the MDA standard. Maximum formation of the MDA-TBA complex was obtained by adjusting the pH between 2.5 and 4.5 and heating the MDA-TBA mixture at 100 degrees C for 60 min. Extraction of the MDA-TBA complex was a critical step and proved complete with n-butanol at pH less than 0.75. It was then evaporated at 37 degrees C under nitrogen. The MDA-TBA complex solubilized in H2O was shown to be stable for at least 7 days. These preparative steps led to the detection of a single peak that on spectral analysis was identified as pure MDA-TBA. This procedure offers several advantages in terms of specificity, recovery, and reproducibility.     

A rapid and preparative method for the separation of yeast ribosomal proteins by using high-performance liquid chromatography.

Ribosomal proteins from the yeast Saccharomyces cerevisiae were separated, on a preparative scale, by ion-exchange h.p.l.c. Proteins from the small and large ribosomal subunits were resolved, respectively, into 33 and 23 peaks, and most of the proteins present in these peaks were identified by using one- and two-dimensional gel electrophoresis. Several of the peaks appeared to contain a single protein uncontaminated by other species. Ribosomal proteins were also separated by using reverse-phase h.p.l.c. Analysis of the peaks resolved indicated that the order of elution for the proteins of both ribosomal subunits is, in certain cases, different for each of the two h.p.l.c. techniques used. Thus a combination of the two chromatographic methods employed here has the potential to facilitate the rapid and preparative separation of each of the proteins present in yeast ribosomes.     

Reverse-phase high-performance liquid chromatography of hydrophobic proteins and fragments thereof

Reverse-phase high-performance liquid chromatography (HPLC) resolution and recovery of cytochrome P-450 and bovine rhodopsin, both integral membrane proteins, and large peptides derived from P-450 LM₂ were enhanced by utilizing ternary solvents. Surprisingly, most test materials eluted later in the gradient when using mixtures of acetonitrile and propanol in the mobile phase compared to using either solvent alone. Of the supports tested, the best recovery of hydrophobic cytochrome P-450 LM₄ was experienced on the less retentive CN-bonded phase. Two alternate solvents for HPLC of polypeptides are proposed: (1) 0.02–0.1 m hexafluoroacetone/NH₃, pH 7.2 for highly acidic peptides; and (2) 6 m formic acid/0.13 m trimethylamine, pH 1.5, vs 4 m formic acid/0.09 m trimethylamine in propanol for relatively insoluble peptides. Anomalous side reactions between formic acid and peptides can cause HPLC peak broadening, increased retention, and decreased resolution. These deleterious effects are thought to be due in part to formyl esterification of serine and threonine residues and appear to be reversible by aminoethanol treatment.     

Anion-exchange chromatography of proteins on AG MP-1 using high-performance liquid chromatography equipment

That the macroporous anion-exchange resin AG MP-1 can be used with HPLC equipment and common aqueous buffers for the chromatography of proteins is shown. The utility of this system is illustrated by the partial purification and complete resolution of the three protein synthesis elongation factors from each other, starting with a crude extract of Escherichia coli. The factors were purified 10- to 30-fold in a yield of 50 to 90% with a single 60-min chromatographic program of increasing NaCl concentration. Other proteins from various biological sources were purified with similar results. Thus, it appears that AG MP-1 is useful, at least in some applications, for the rapid, reproducible, and economical purification of proteins using HPLC equipment.     

Purification of liver and hepatoma membrane proteins by high-performance liquid chromatography

This paper documents the recovery of selected proteins from hepatic plasma membranes. Initial purification, achieved by a series of stepwise extractions, facilitates the subsequent purification by HPLC. Examples are provided to illustrate the recovery of specific proteins from two Morris hepatoma lines and the liver.     

Rapid separation of gonadotropin-releasing hormone molecular forms by isocratic high-performance liquid chromatography on an ion-exchange column

The purpose of the present work was to develop a chromatographic system for the separation of five molecular forms of the gonadotropin-releasing hormone (GnRH); mammalian GnRH (mGnRH) (LHRH), salmon GnRH (sGnRH), chicken I GnRH (clGnRH), chicken II GnRH (cIIGnRH) and lamprey GnRH I (IGnRH-I). By using an ion-exchange HPLC column and isocratic elution, it was possible to separate properly the five peptides in approximately 20 min. The utility of the system in determining the GnRHs forms present in the brain of two species of vertebrates was examined.