Fast Protein Liquid Chromatography System Research Articles

Separation and characterization of high-density lipoprotein subpopulations by gel permeation chromatography

High-density lipoproteins (HDL) contain at least five distinct subpopulations when analyzed by gradient gel electrophoresis. This report represents the first description of a simple technique for isolating these subpopulations of HDL in quantities sufficient to enable characterization in terms of particle size, apolipoprotein AI and apolipoprotein AII content and chemical composition. Lipoproteins were separated and subfractionated on a column of Superose 6B using a fast protein liquid chromatography system. Five normal subjects were studied: HDL 2b and HDL 3a were isolated as essentially single subpopulations from all subjects, while HDL 2a could be isolated from only three of the subjects. HDL 3b was isolated in a relatively impure from (70%) from all subjects. Identical subpopulations were identified in each subject by gradient gel electrophoresis of unseparated HDL.

Fast protein affinity chromatography of two flavonoid glucosyltransferases

Chrysosplenium americanum contains a glucosyltransferase which attacks the 2′- or 5′-positions of partially methylated flavonols. The two enzyme activities were not resolved on either Superose 12 (gel filtration), Mono Q (ion exchange) or Mono P (chromatofocusing) columns, suggesting similar chromatographic properties. The combined use of the affinity columns UDP-glucuronic acid agarose and Reactive Brown 10-agarose, coupled with a fast protein liquid chromatography system allowed the separation, for the first time, of the two flavonol-ring B-specific glucosyltransferases.

Identification of different molecular forms of human airway lysozyme

Human airway lysozyme (HAL) was separated into fractions of distinct molecular forms using a Mono S cation-exchange column on a fast-protein liquid chromatography system. This new and rapid (30 min) purification procedure of human lysozyme enabled the preparation of fractions, highly enriched in different isoenzymes of HAL. Purified HAL from (i) pathological purulent airway secretions, (ii) nonpurulent airway secretions, and (iii) normal tracheobronchial tissue culture medium was characterized by four, three, and only one enzymatically active molecular forms, respectively. All charge forms (separated or combined) recovered from either purulent or nonpurulent airway secretions or tracheobronchial culture medium exhibited the same apparent molecular weight of 15,000.

Chromatofocusing of mammalian estrone sulfate sulfohydrolase activity

Estrone sulfate sulfohydrolase (estrogen sulfatase) activity was solubilized by treatment with Triton X-100 from 105,000 g pellets of guinea pig uterus, testis and brain, as well as from rat liver and human placenta. The solubilized forms were subjected to chromatofocusing in the fast protein liquid chromatography (FPLC) system and on conventional columns packed in our laboratory. The guinea pig tissue pattern was complex. Uterus showed peaks of activity with apparent pI's of 9.11 and 7.6; testis contained 3 peaks with pI's of 9.18, 8.7 and 7.5; brain possessed peaks with pI's of 9.28 and 8.6. In each case the major activity peak was that with pI > 9. Rat liver activity chromatofocused as a single peak of apparent pI = 6.87 and the human placental enzyme also showed a single, though broad, peak, of pI = 6.57. This suggests not only that the guinea pig enzyme(s) differs markedly from those of rat liver and human placenta, but that there may be qualitative differences between the forms in the three guinea pig tissues. Chromatofocusing behaviour was not independent of the specific exchange resins and ampholytes utilized. The recovered enzyme activity was fairly stable and it seems that chromatofocusing could be a useful step in purification of the guinea pig enzyme(s), particularly the main form possessing a pI > 9.

The rapid purification and partial characterization of human sperm proacrosin using an automated fast protein liquid chromatography (FPLC) system

A rapid efficient procedure was developed for obtaining highly purified human proacrosin. Ejaculated spermatozoa were washed via centrifugation through 1 M sucrose containing 50 mM benzamidine and acid-extracted in the presence of benzamidine. The solubilized material was dialyzed then lyophilized. The sample was resuspended in 8 M guanidine hydrochloride in acetic acid (0.5 M) pH 2.5 and then subjected to gel permeation chromatography with an automated fast protein liquid chromatography system utilizing two Pharmacia Superose 12 columns set in tandem that were equilibrated in the same buffer. The proacrosin eluted as an individual peak that was well separated from another proteinase zymogen referred to as sperminogen. The proacrosin preparation was determined to be highly purified when observed on silverstained SDS-polyacrylamide gels as well as on gelatin-SDS-polyacrylamide gels. The proacrosin appeared as a doublet ( M r = 55 000 and 53 000) on both of these systems. The autoconversion of proacrosin to acrosin at pH 8 resulted in a typical sigmoidal autoactivation curve. Following protein staining of SDS-polyacrylamide gels, it was shown that upon activation of purified proacrosin preparations the 55 000 and 53 000 molecular weight proteins were initially degraded to a 49 000 form and then to several lower molecular weight forms ( M r = 40 000 – 34 000). Similar findings with regard to proteolytic digestion were observed following gelatin-SDS-polyacrylamide zymography except that an increase with time in proteinase intensity between 58 000 and 53 000 was also observed. Cobalt and calcium were found to be potent inhibitors of the conversion of proacrosin into acrosin, while sodium resulted in much less inhibition of this process. Calcium was found to markedly enhance the proteolytic activity of human acrosin, while it had no observable influence on the acrosin hydrolysis of benzoylarginine ethyl ester. Thus, the described purification procedure resulted in a highly purified proacrosin preparation in sufficient yields to allow for its partial characterization.

Rapid separation of Escherichia coli 30S ribosomal proteins by fast protein liquid chromatography (FPLC)

The proteins of the 30S ribosomal subunit from Escherichia coli have been separated by reverse-phase high-performance liquid chromatography on a short alkyl chain (C1/C8)-coated phase. The reverse-phase column was connected to a fast protein liquid chromatography (FPLC) system. The 21 proteins of the 30S ribosomal subunit were resolved into 16 peaks. Eleven proteins were isolated in purified form in a single chromatographic run as shown by polyacrylamide gel electrophoresis and amino acid analysis. Interestingly, the retention times of some proteins differed from the retention times observed on other reversed-phase support materials. The results show the speed and resolution of reverse-phase FPLC for both analytical and semi-preparative separations of 30S ribosomal proteins.

Rapid fractionation of collagen chains and peptides by high-performance liquid chromatography

A strategy was developed, using a Pharmacia Fast Protein Liquid chromatography (FPLC) system, for the rapid preparation of the α-chains, cyanogen bromide peptides and tryptic peptides of type I collagen obtained from tissues and cultured fibroblasts. Collagen α-chains were prepared using a C 18 PEP-RPC reverse-phase column and volatile solvents. Preliminary Superose 6 gel permeation chromatography was used to separate the crosslinked β- and γ-chains from the α-chains of tissue collagen samples. A Mono S cation-exchange column was used to resolve all of the major type I collagen cyanogen bromide peptides including the α1(I)CB7 and CB8 peptides, which have not been well resolved by previously published methods. Collagen tryptic peptides were chromatographed on the PEP-RPC reverse-phase column.

Separation of parasite antigens by molecular exclusion, anion exchange, and chromatofocusing utilizing FPLC protein fractionation systems

Excretory-secretory products (ESP) were harvested from balanced salt solutions in which adult Fasciola hepatica had been incubated for 4–6 h at 37°C. The ESP was fractionated by standard low pressure molecular exclusion chromatography and FPLC (fast protein liquid chromatography) using the principles of molecular exclusion, anion exchange, and chromatofocusing. The dot-enzyme-linked immunosorbent assay (Dot- ELISA) was used to demonstrate the immunoreactivity of eluted fractions. Compared to Sephacryl S-200, separation by Superose-6 (FPLC) was faster and resolved more peaks (four with Sephacryl S-200 and nine with Superose-6). Peaks from Sephacryl S-200 were resolved by the first anion exchange (Mono Q) separation into seven peaks; when these peaks were subjected to a second anion exchange, 15 peaks were resolved. Thirty-eight peaks were resolved by chromatofocusing (Mono P) in the pH range 7-4. Immunoreactive fractions from narrow-range (single pH unit) chromatofocusing were identified by the Dot-ELISA. The FPLC system proved to be a means of rapid and high resolution separation of F. hepatica antigens.

Small-column chromatofocusing of cerebrospinal fluid and serum immunoglobulin G

Chromatofocusing performed by the Pharmacia fast protein liquid chromatographic system equipped with a specially designed small column was applied for examinations of submilligram quantities of cerebrospinal fluid and serum immunoglobulin G. The separations were based on a pH gradient between 9.5 and 6.0. Mono- or oligoclonal immunoglobulin G components having p I values within the optimal working range of the gradient were easily identified and the findings differed clearly from those of normal immunoglobulin G. The capacity to detect abnormal immunoglobulin G components compared well with previous experiences from the commercially available Mono P column. The small column offers advantages by having a shorter separation time, a decreased dilution of sample in the eluate and a lower consumption of start and eluent buffers.

Purification of human plasma lipid transfer protein using fast protein liquid chromatography

A system for the isolation of human plasma lipid transfer protein (LTP) has been devised using a combination of conventional and high-performance ion-exchange chromatography. Following initial purification by ammonium sulphate precipitation, ultracentrifugation, hydrophobic interaction and cation-exchange chromatography, appropriate fractions were further purified using the Pharmacia fast protein liquid chromatography system. Using this method of purification, human plasma LTP has been purified more rapidly and with greater recovery than with conventional column chromatography. Whereas two forms of LTP were previously reported from the authors' laboratory [LTP-I, molecular mass ( M r) 69 000 and LTP-II, M r 55 000], with an improved chromatographic system only one form of LTP (LTP-I) has been isolated. This suggests that LTP-II may have been a fragment of LTP-I, produced during the previously used lengthy purification process.

Tandem purification of IgM monoclonal antibodies from mouse ascites fluids by anion-exchange and gel fast protein liquid chromatography

A tandem chromatographic procedure was used to isolate rapidly mouse IgM monoclonal antibodies. Mouse ascites fluids containing IgM monoclonal antibodies were first chromatographed on an anion-exchange Mono Q column connected to a fast protein liquid chromatography system. The IgM-rich fractions frm the Mno Q column were then injected on a gel filtration Superose 6 column equilibrated with a low-ionic stength buffer and eluted with a high-ionic stregth buffer. Assessment of the purity of isolated IgM monoclonal antibodies was performed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis together with a Coomassie Brilliant Blue R 250 staining technique. Assesment of the immunoreactivity of isolated IgM monoclonal antibodies was performed by a enzyme linked inmunosorbent assay using a solid phase adsorbed antigen against which IgM monoclonal antibodies were directed. The chromatographic procedure described provides a new method for the rapid purification of mouse IgM monoclonal antibodies to a high degree of purity and in a immunoreactive state.

Tandem purification of mouse IgM monoclonal atibodies produced in vitro using anion-exchange and gel fast protein liquid chromatography

A tande chromatographic procedure was used to isolate rapidly mouse IgM monclonal antibodies produced by cultivation of hybridomas in vitro. Hybridoma culture supernatants containing mouse IgM monoclonal antibodies were first chromatographed on an anion-exchange Mono Q column connected to a fast protein liquid chromatography system. This anion-exchange step offers the advantage of obtaining IgM antibodies in a concentrated form The IgM-rich fractions from the Mono Q column were then injected on a gel filtration Superose 6 column equilibrated with a low-ionic strength buffer and eluted with a high-ionic strength buffer. Assessment of the purity of isolated IgM monoclonal antibodies was performed by sodium dodecyl sulphate polyacrylamide gel electrophoresis together with a Coomassie Brillant Blue r 250 staining technique. Assessment of the immunoreactivity of isolated IgM monoclonal antibodies was performed by an enzyme linked immunosorbent assay using a solid phase adsorbed antigen against which IgM monoclonal antibodies were directed. The chromatographic procedures described allows the rapid isolation of mouse IgM monoclonal antibodies produced in vitro at a high degree of purity and in an immunoreactive state.

Rapid Separation of Milk Whey Proteins by Anion Exchange Chromatography

A fast protein liquid chromatography system was used to fractionate the major proteins of sweet and acid wheys. Fifty to 500μl of whey were fractionated with a stepwise ionic strength gradient using water (buffer A) and increasing concentrations of .7 M sodium acetate (buffer B). Six well-resolved peaks were obtained: 1) amino acids (tentative identification), 2) low molecular weight peptides (tentative identification), 3) highly enriched α-lactalbumin, 4) highly enriched serum albumin 5) electrophoretically pure β-lactoglobulin B, and electrophoretically pure β-lactoglobulin A. A poor baseline or unresolved peaks resulted when .02 M bis-tris or .02 M histidine was used for buffer A and .7 M sodium acetate in .02 M bis-tris or histidine was used for buffer B. When sodium chloride was used in place of sodium acetate, β-lactoglobulins A and B were poorly resolved. The column was cleaned after each run by injecting 2ml of the following reagents: glacial acetic acid, 2N sodium chloride, 2N sodium hydroxide, 2N sodium chloride, 2% detergent, and 100% acetonitrile. Time required to run each sample including column cleanup was 40min.

Improved enzyme screening by automated fast protein liquid chromatography

The assay for NADH-dependent dehydrogenases in crude extracts is often interfered with nonspecific reactions. Therefore a screening for such enzymes is hampered by high blank values. To overcome such problems we chromatographed crude extracts on a fast protein liquid chromatography system during part of an enzyme screening for 2-hydroxyisocaproate dehydrogenases and lactate dehydrogenases. The automated chromatography procedure presented consists of a combination of gel filtration and ion-exchange chromatography. The total time needed to perform one cycle of the two-column purification, including the equilibration and regeneration steps, is about 35 min. The procedure described separates the desired enzyme, 2-hydroxyisocaproate dehydrogenase, totally from any interfering activity such as NADH-oxidase and also from the second enzyme of interest, the lactate dehydrogenase. Besides the elimination of the side reactions the desired enzymes are purified up to 20-fold.

Studies of the heterogeneity of human pituitary LH by fast protein liquid chromatography.

The Pharmacia fast protein liquid chromatography system was employed to fractionate a purified preparation of human LH (hLH) on the anion exchanger Mono Q at pH 7 X 8 into 14 sub-fractions. Each of the sub-fractions was characterized by its behaviour on polyacrylamide gel electrophoresis, sodium dodecyl sulphate (SDS) gel electrophoresis, LH receptor binding activity and sialic acid content. All sub-fractions contained sialic acid, were active in binding to LH receptors, and exhibited components typical of hLH subunits on SDS gel electrophoresis. None of the subfractions was homogeneous with respect to charge. There is evidence that part of the heterogeneity results from the presence in some molecules of an internal proteolytic cleavage within the beta-subunit, and fractions enriched in species containing such cleavages were prepared by this method.

Purification of a uridine-specific acid nuclease from chicken liver by fast protein liquid chromatography

A rapid purification for a novel uridine-specific nuclease from chicken liver based on the Pharmacia fast protein liquid chromatography (FPLC) system is presented. The purification was achieved by applying crude extract to a Mono S cation-exchange column equilibrated with 10 m M potassium phosphate buffer (pH 6.0). The enzyme was eluted in 20 min with a potassium chloride gradient at a flowrate of 2 ml/min. The enzyme was then chromatographed on a Superose 12 size-exclusion column in less than 1 h at a flow-rate of 0.5 ml/min ( K av = 0.77). The enzyme was re-chromatographed on a second Mono S column to concentrate the protein. The uridine-specific nuclease hydrolyzed poly(U) and Escherichia coli 5S RNA. Poly(A) was hydrolyzed by the nuclease less efficiently (about 10% of the poly(U) activity). No hydrolysis was detected with poly(C), poly(G), poly(dT) or poly(dA) as substrate. The speed with which each purification step could be carried out facilitated the determination of optimal chromatographic conditions. We found that the resolution of the Mono S and Superose 12 columns was superior to that of conventional ion exchangers and size-exclusion columns respectively.

Determination of the R-protein and the R-protein-vitamin B12-complex in saliva and gastrointestinal juice by FPLC Mono S cationic chromatography.

The binding of vitamin B12 to the two vitamin B12 binding proteins--intrinsic factor and R-protein--in gastrointestinal juice is pH dependent. It is therefore of importance that binding studies are carried out at pH values near the physiological pH of the gastric juice. In the present study the vitamin B12 complexes of the two vitamin B12 binders were separated at a pH of 1.8 using the cationic exchange chromatograph Mono S attached to the fast protein liquid chromatography (FPLC) system. The R-protein concentration was measured in saliva and gastric juice with high accuracy and with highly significant correlation compared to the serum-coated charcoal method of Gottlieb. The method showed a decreased recovery in duodenal juice and in samples with high bile content.

Fractionation of polyclonal antibody by isoelectric focusing and chromatofocusing: Separation of high-affinity rabbit clonotype anti-thyroxine antibody

Immunogobulin G fractions prepared from conventional rabbit anti-thyroxine (T 4) antisera were fractionated by agarose gel isoelectric focusing (IEF) in the range of pH 3 to 10, and by chromatofocusing using a Fast Protein Liquid Chromatography (FPLC) system. The clonotype antibodies were recovered from the fractions and subjected to Scatchard plot anlysis. The highest affinity constants of the initial antibody (shown in parentheses) and those of the antibodies recovered were IEF, 1.8 × 10 9 to 8.3 × 10 9 m −1(2.2 × 10 9 m −1); FPLC, 2.4 × 10 9 to 6.0 × 10 9 m −1 (2.5 × 10 9 m −1). A sensitive radioimmunoassay of T 4 was achieved with the isolated high-affinity anti-T 4 antibody. The minimum detectable concentration of T 4 was 6.3 × 10 −15 to 1.5 × 10 −14 mol/tube, which was three to five times lower than that detectable with the initial antibodies.

Some properties of brain opioid receptors in membrane bound and solubilized state

In brain membrane preparations, 3H-naloxone binding to bullfrog brain gave the highest values versus other species such as carp, chicken, rat and mouse. Scatchard analysis revealed that the Bmax value of bullfrog brain membranes was three fold greater than that of rat brain membranes. Opioid receptors were solubilized from bullfrog brain membranes using digitonin or 3-(cholamidopropyl)-dimethylammonio-1-propane sulfonate (CHAPS) as the detergent. The properties of solubilized opioid receptors were essentially the same as membrane bound receptors with regard to sensitivity to various treatments. The order of potency for a series of agonists in displacing 3H-naloxone binding in both membrane and solubilized preparations was bremazocine > pentazocine > morphine > δ-receptor peptide. Partial purification of opioid receptors was performed using a fast protein liquid chromatography system. Specific binding activity of 3H-naloxone was increased 10–15 fold within 20 min using the Mono Q column.

Use of fast protein liquid chromatography in the purification of inhibin from bovine follicular fluid

Inhibin from bovine follicular fluid was partly purified using affinity chromatography on immobilized Procion Red 3B, gel filtration on Sephadex G-25 and ion-exchange chromatography on the fast protein liquid chromatography system. Inhibin was subsequently characterized using preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroelution. Biological activity was associated with a protein with an apparent molecular weight of approximately 65 kD.