Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.     

Detection and characterization of lipid hydroperoxides at picomole levels by high-performance liquid chromatography

A new method for the detection of various lipid hydroperoxides and hydrogen peroxide at the picomole level has been developed by combining an HPLC system with an ultrasensitive analytical system based on the detection of chemiluminescence emitted by isoluminol in the presence of hydroperoxide and microperoxidase. This HPLC separation removes interfering antioxidants so that the method can be applied to biological samples such as blood plasma lipids. Several HPLC conditions are described which allow simple identification of different lipid hydroperoxides.     

Composition of the ternary protein complex of the red cell membrane cytoskeleton

The red cell membrane skeletal network is constructed from actin, spectrin and protein 4.1 in a molar ratio of actin subunits/spectrin heterodimer/protein 4.1 of 2:1:1. This represents saturation of the actin filaments, since incubation with extraneous spectrin and protein 4.1 leads to no binding of additional spectrin, either to the inner surface of ghost membranes or to lipid-free membrane cytoskeletons. Partial extraction of spectrin from the membrane is accompanied by release of actin under all conditions. Regardless of the proportion of spectrin extracted, the molar ratio of spectrin dimers/actin subunits is constant at 1:2. This is not the result of release or cooperative breakdown of whole lattice junctions from the network, for the number of actin filaments, judged by capacity to nucleate polymerisation of added G-actin, remains unchanged even when as much as 60% of the total spectrin has been lost. A similar 1:2:1 stoichiometry characterises the complex formed when G-actin is allowed to polymerise in the presence of varying amounts of spectrin and protein 4.1. When this complex is treated with the depolymerising agent, 1 M guanidine hydrochloride, it breaks down into smaller units of the same stoichiometry. After cross-linking these can be recovered from a gel-filtration column. Complexes prepared starting from G-actin appear to be much more stable than those formed when spectrin and protein 4.1 are bound to F-actin.     

Substructure of human erythrocyte spectrin

The human erythrocyte structural protein spectrin and its subunits I, II were isolated in the presence of Na-dodecyl-sulfate by gel filtration and preparative gel electrophoresis. After removal of the detergent, spectrin alpha-helical content is comparable to spectrin isolated without detergent. Subunits I and II formed single bands in isoelectric focusing (pI = 5.6) and in Ornstein-Davis disc gel electrophoresis systems, indicating the individual subunits are homogenous in nature. The molecular weights of the subunits I and II, determined by Ferguson plot, are 237,500 and 238,600, respectively, which is in good agreement with values obtained by the standard SDS gel relative mobility method. Limited tryptic digestion of spectrin and two-dimensional peptide maps of the individual subunits cleaved by S-cyanylation reaction showed dissimilar patterns, suggesting differences in primary structure between the two subunits.     

Drosophilia spectrin. I. Characterization of the purified protein

We purified a protein from Drosophila S3 tissue culture cells that has many of the diagnostic features of spectrin from vertebrate organisms: (a) The protein consists of two equimolar subunits (Mr = 234 and 226 kD) that can be reversibly cross-linked into a complex composed of equal amounts of the two subunits. (b) Electron microscopy of the native molecule reveals two intertwined, elongated strands with a contour length of 180 nm. (c) Antibodies directed against vertebrate spectrin react with the Drosophila protein and, similarly, antibodies to the Drosophila protein react with vertebrate spectrins. One monoclonal antibody has been found to react with both of the Drosophila subunits and with both subunits of vertebrate brain spectrin. (d) The Drosophila protein exhibits both actin-binding and calcium-dependent calmodulin-binding activities. Based on the above criteria, this protein appears to be a bona fide member of the spectrin family of proteins.     

Universal calibration of gel permeation chromatography and determination of molecular shape in solution

Gel permeation chromatography (GPC) has become a routine technique for both biology and polymer chemistry. By comparison our theoretical perception of the separation principle of GPC is still immature and conflicting and so is the assessment of the analytical informational content of this method. In order to discriminate between the various parameters that might influence GPC and thus to decide among the numerous propositions of calibration, several odd biopolymers (tropomyosin, spectrin, DNA, tobacco mosaic virus, alpha-actinin, ovomucoid) were selected. They were characterized by analytical ultracentrifugation as well as quasielastic light scattering, and they were compared to globular proteins including icosahedral viruses (tomato bushy stunt virus, turnip yellow mosaic virus, Q beta, MS2) on several different HPLC column matrices. The results demonstrate that the universal calibration principle of GPC is the viscosity radius, i.e., the molecular volume times a shape function which is defined by the intrinsic viscosity. Alternate propositions such as molecular weight, second virial coefficient, diffusion coefficient (Stokes radius), radius of gyration, mean linear projected length, contour length, and related measures seem to be excluded on the basis of the evidence presented. These results help to focus the physical picture which seems to govern GPC. Finally it is demonstrated that GPC is a versatile and unique tool with which to characterize molecular shape and dynamics in solution.     

Analysis of 5-pyrrolidone-2-carboxylate ester by reverse phase high-performance liquid chromatography

A simple quantitative method for estimating nanomole concentrations of 5-pyrrolidone-2-carboxylic acid (PCA) in tissue homogenates from mouse has been developed using reverse-phase HPLC. PCA was detected as the 4-nitrophenacyl ester which has an absorption maximum at 263 nm, a relatively high stability, and excellent chromatographic separation and detectability. This method offers distinct advantages over other analytical procedures thus far employed for measuring PCA in that the 4-nitrophenacyl derivative of PCA can be readily prepared from deproteinized tissue homogenates and quantitated by HPLC within relatively short time intervals with good precision and specificity.     

Spectrin synthesis in the preimplantation mouse embryo

The preimplantation mouse embryo expresses two polypeptides, Mr 240,000 and Mr 235,000, that are immunologically cross-reactive with antibody to the alpha and beta subunits of mouse brain spectrin. We investigated the synthesis of the spectrin subunits in the Triton-soluble and Triton-insoluble fractions of fertilized eggs, two-cell embryos, compacted morulae, and blastocysts labeled with L-[35S]methionine. Synthesis of embryonic spectrin began in the Triton-soluble fraction with significant levels of alpha-spectrin synthesis first detected in the morula stage and significant levels of beta-spectrin synthesis detected in the blastocyst stage. Incorporation of newly synthesized alpha- and beta-spectrin into the cytoskeletal fraction took place in the blastocyst when equal amounts of both subunits were assembled. Previous studies have shown Triton-insoluble spectrin to be concentrated in regions of cell-cell contact in the embryo (J. S. Sobel and M. A. Alliegro, 1985, J. Cell Biol. 100, 333-336). The temporal and spatial correlation between the assembly of newly synthesized spectrin and its concentration in regions of cell apposition is consistent with the hypothesis that cell contact may influence the assembly of embryonic spectrin.     

Separation and purification of sphingomyelin diastereomers by high-performance liquid chromatography

All naturally occurring sphingomyelins have the d-erythro-(2S,3R) configuration of the sphingoid base. We have developed a normal-phase HPLC method for the separation of this natural stereoisomer from the l-threo-sphingomyelin, which is the other stereoisomer commonly present in semisynthetic preparations of acyl-chain defined sphingomyelins. The chromatographic method was developed by modification of a previously reported method for phospholipid separation on a normal-phase diol column. The separation was accomplished by a binary gradient of solvent mixtures (A) hexane:isopropanol:acetic acid (82:17:1.0 by vol) and (B) isopropanol:water:acetic acid (85:14:1.0 by vol) with 0.08 vol% triethylamine added to both solvent mixtures. The program of gradient elution was optimized for maximal separation of sphingomyelin diastereomers. For detection of the lipids, a light-scattering detector was used. This analytical scale HPLC method was also used for purification of the stereoisomers (up to 0.5 mg of N-oleoyl-sphingomyelin in a single injection). The purified stereoisomers were at least 99% pure according to high-performance thin-layer chromatography and analytical HPLC.     

Separation of nucleoside phosphoramidate diastereoisomers by high performance liquid chromatography and capillary electrophoresis

Separations of five diastereoisomers of nucleoside phosphoramidate derivatives (pronucleotides) were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and CE method using anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using in a normal-phase methodology. Capillary electrophoresis was used as an alternative technique to HPLC for the separation of pronucleotides. The diastereoisomers were fully resolved with sulfated cyclodextrins at both BGE pH (2.5 and 6.2). Limits of detection and limits of quantification, calculated for both methods, are up to 200 times higher in CE separations than in HPLC separations. The analytical HPLC method was then applied in a preliminary study for the pronucleotide 1 quantification in cellular extract.     

Calmodulin inhibits the phosphorylation of spectrin in vitro

In vitro phosphorylation of purified spectrin dimer was studied in the presence of Ca2+-calmodulin (CaM). CaM inhibited autophosphorylation of the beta subunit of spectrin. The inhibitory effect (65% at a 32-fold molar excess) appeared to be due to a weak interaction of CaM with spectrin. CaM was similarly effective in a phosphatase-stimulated autothiophosphorylation of the beta subunit with [gamma-35S]ATP. Hence, its inhibitory effect was not due to stimulation of a spectrin-associated phosphatase activity. Phosphorylation of spectrin by the catalytic subunit of a cAMP-dependent protein kinase occurred in both subunits (1984, FEBS Lett. 169, 323). CaM selectively inhibited a cAMP-dependent phosphorylation of the alpha subunit of spectrin to 30% at two CaM per spectrin. It was ineffective on the cAMP-dependent phosphorylation of the beta subunit up to a 32-fold molar excess. These results yield functional evidence for a CaM-spectrin interaction. They further suggest that CaM can regulate the extent of a cAMP-dependent phosphorylation of the alpha subunit of spectrin.     

Contributions of the beta-subunit to spectrin structure and function

The three avian spectrins that have been characterized consist of a common alpha-subunit (240 kD) paired with an isoform-specific beta-subunit from either erythrocyte (220 or 230 kD), brain (235 kD), or intestinal brush border (260 kD). Analysis of avian spectrins, with their naturally occurring "subunit replacement" has proved useful in assessing the relative contribution of each subunit to spectrin function. In this study we have completed a survey of avian spectrin binding properties and present morphometric analysis of the relative flexibility and linearity of various avian and human spectrin isoforms. Evidence is presented that, like its mammalian counterpart, avian brain spectrin binds human erythroid ankyrin with low affinity. Cosedimentation analysis demonstrates that 1) avian erythroid protein 4.1 stimulates spectrin-actin binding of both mammalian and avian erythrocyte and brain spectrins, but not the TW 260/240 isoform, 2) calpactin I does not potentiate actin binding of either TW 260/240 or brain spectrin, and 3) erythrocyte adducin does not stimulate the interaction of TW 260/240 with actin. In addition, a morphometric analysis of rotary-shadow images of spectrin isoforms, individual subunits, and reconstituted complexes from isolated subunits was performed. This analysis revealed that the overall flexibility and linearity of a given spectrin heterodimer and tetramer is largely determined by the intrinsic rigidity and linearity of its beta-spectrin subunit. No additional rigidity appears to be imparted by noncovalent associations between the subunits. The scaled flexural rigidity of the most rigid spectrin analyzed (human brain) is similar to that reported for F-actin.     

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.     

Isolation of spectrin subunits and reassociation in vitro. Analysis by fluorescence polarization

Fluorescence polarization has been used to probe the exposure of tryptophan residues of erythrocyte spectrin. A significant decrease in anisotropy occurred when spectrin was heated at temperatures ranging from 38 to 48 degrees C. At low concentrations of urea, these anisotropy changes shifted to lower temperatures and were minimal in concentrations of urea 3 M or greater. These findings were attributed to the stepwise unfolding of the subdomain structure of spectrin under these conditions and eventual dissociation of oligomeric spectrin to the monomer state. DEAE-cellulose column chromatography in the presence of 3 M urea confirmed this prediction and permitted isolation of pure alpha and beta subunits of spectrin in good yields. The isolated subunits were soluble in neutral salt solutions and were readily reconstituted into high molecular weight forms that displayed "native" tryptophan fluorescence anisotropy changes and migrated as discrete oligomeric species when analyzed by nondenaturing acrylamide gel electrophoresis. The reconstituted complexes were indistinguishable from native spectrin molecules when examined by low angle shadowing and electron microscopy.     

Improving LC-MS sensitivity through increases in chromatographic performance: comparisons of UPLC-ES/MS/MS to HPLC-ES/MS/MS

Recent technological advances have made available reverse phase chromatographic media with a 1.7 microm particle size along with a liquid handling system that can operate such columns at much higher pressures. This technology, termed ultra performance liquid chromatography (UPLC), offers significant theoretical advantages in resolution, speed, and sensitivity for analytical determinations, particularly when coupled with mass spectrometers capable of high-speed acquisitions. This paper explores the differences in LC-MS performance by conducting a side-by-side comparison of UPLC for several methods previously optimized for HPLC-based separation and quantification of multiple analytes with maximum throughput. In general, UPLC produced significant improvements in method sensitivity, speed, and resolution. Sensitivity increases with UPLC, which were found to be analyte-dependent, were as large as 10-fold and improvements in method speed were as large as 5-fold under conditions of comparable peak separations. Improvements in chromatographic resolution with UPLC were apparent from generally narrower peak widths and from a separation of diastereomers not possible using HPLC. Overall, the improvements in LC-MS method sensitivity, speed, and resolution provided by UPLC show that further advances can be made in analytical methodology to add significant value to hypothesis-driven research.     

Subunit analysis of mitochondrial cytochrome c oxidase and cytochrome bc1 by reversed-phase high-performance liquid chromatography

A rapid separation of the ten nuclearly-encoded subunits of mitochondrial cytochrome c oxidase, and ten out of the eleven subunits of cytochrome bc₁, was achieved using a short, 50 mm C₁₈-reversed-phase column. The short column decreased the elution time 4–7 fold while maintaining the same resolution quality. Elution was similar to a previously published protocol, i.e., a water/acetonitrile elution gradient containing trifluoroacetic acid. Isolated subunits were identified by MALDI-TOF. The rapidity of the described method makes it extremely useful for determining the subunit composition of isolated mitochondrial complexes. The method can be used for both analytical and micro-preparative purposes.     

Two-dimensional separation of erythrocyte membrane proteins.

The details of a two-dimensional separation procedure specially designed for the study of erythrocyte membranes are presented. In this highly reproducible method, the membrane proteins are dissolved in sodium dodecyl sulfate and separated first on the basis of charge by isoelectric focusing. The samples are loaded either at the cathode (CIF) or anode (AIF). The CIF samples gave better separation of the acidic proteins, while the AIF was better for the separation of the high molecular weight polypeptides of the erythrocyte. Over 90 discrete polypeptides could be detected with this method in the pH range of 5 to 8. Special attention was given to the higher molecular weight components. For example, six components could be detected within the 90,000 to 100,000 molecular weight range of protein 3, the major membrane protein. A component with the same or very nearly the same molecular weight as spectrin band 2 was detected. It is more basic than spectrin band 2, and both spectrin band 2 and the basic component are readily phosphorylated in the intact cell. However, the phosphorylation of band 2 is cAMP independent while the phosphorylation of the basic component is enhanced by cAMP. In contrast to spectrin, the basic component is not extracted from the membrane with 0.1 mm EDTA, although dilute NaOH will remove it from the membrane. The Ca²⁺-activated transferase of the erythrocyte cytoplasm will not crosslink this component. Calcium does, however, activate the conversion of this component to a lower molecular weight. This high molecular weight basic component has properties attributed to the component labeled 2.1 in Fairbanks' system of nomenclature.     

Determination of 5-hydroxytryptamine metabolites in isolated perfused rabbit lung by high-performance liquid chromatography.

An analytical procedure, utilizing high-performance liquid chromatography (HPLC) hasbeen developed for the separation of radiolabeled metabolites of 5-hydroxytryptamine (5-HT) in biological fluids. Four different chromatographic systems are described, which enable the separation of groups of metabolites possessing similar organic functionality to be achieved. As an example of this general analytical method, it is demonstrated that no methylation of 5-HT occurs in perfused rabbit lung, the principal metabolites being 5-hydroxyindoleacetic acid and 5-hydroxytryptophol.     

Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization-tandem mass spectrometry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 μg protein) or preparative (>250 μg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.     

Beta spectrin bestows protein 4.1 sensitivity on spectrin-actin interactions

The ability of protein 4.1 to stimulate the binding of spectrin to F-actin has been compared by cosedimentation analysis for three avian (erythrocyte, brain, and brush border) and two mammalian (erythrocyte and brain) spectrin isoforms. Human erythroid protein 4.1 stimulated actin binding of all spectrins except the brush border isoform (TW 260/240). These results suggested that the beta subunit determined the protein 4.1 sensitivity of the heterodimer, since all avian alpha subunits are encoded by a single gene. Tissue-specific posttranslational modification of the alpha subunit was excluded by examining the properties of hybrid spectrins composed of the purified alpha subunit from avian erythrocyte or brush border spectrin and the beta subunit of human erythrocyte spectrin. A hybrid composed of avian brush border alpha and human erythroid beta spectrin ran on nondenaturing gels as a discrete band, migrating near human erythroid spectrin tetramers. The actin-binding activity of this hybrid was stimulated by protein 4.1, while either chain alone was devoid of activity. Therefore, although both subunits were required for actin binding, the sensitivity of the spectrin-actin interaction to protein 4.1 is a property uniquely bestowed on the heterodimer by the beta subunit. The singular insensitivity of brush border spectrin to stimulation by erythroid protein 4.1 was also consistent with the absence of proteins in avian intestinal epithelial cells which were immunoreactive with polyclonal antisera sensitive to all of the known avian and human erythroid 4.1 isoforms.