Chromatographic Heterogeneity Research Articles

Acid phosphatases and ribonucleases from Dactylis glomerata seeds. I. Chromatographic and electrophoretic heterogeneity of the enzymes

Acid phosphatase and ribonuclease extracted with 0.1 M sodium acetate buffer, pH 5.1 from Dactylis glomerata seeds, and partially purified by means of 70% ethanol precipitation showed electrophoretic and Chromatographic heterogeneity. After chromatography on DEAE-cellulose acid phosphatase and ribonuclease were separated into four peaks. Nonadsorbing acid phosphatase on DEAE-cellulose (peak I) was separated into four peaks on CM-cellulose. The highest activity (11 units/mg) was found in fraction b (acid phosphatase Ib). The enzyme was activated by Mg<sup>2+</sup>, Ca<sup>2+</sup>, Li<sup>+</sup>, Cs<sup>+</sup>, K<sup>+</sup> ions and inhibited by Cu<sup>2+</sup>, Zu<sup>2+</sup>, F<sup>-</sup> and Mo<sup>-6</sup> at optimum pH 5.0. Strong absorbing ribonuclease on DEAE-cellulose (peak IV) was further separated on G-200 Sephadex into two molecular forms: RN-asa1 and RN-ase2. Ribonuclease l, a thermolabile enzyme with specific activity 807 units/mg, showed an optimal activity at pH 4.8-5.1.

LH pulsatility, biopotency, and clearance during undernutrition in orchidectomized mature rats.

The effect of food restriction on circulating luteinizing hormone (LH) levels in orchidectomized rats is controversial. The present study demonstrates that decreasing food intake by 50% for 3-10 days in orchidectomized rats increases LH pulse amplitude, length, area under pulse curve, and mean levels but decreases LH pulse frequency compared with ad-lib fed, orchidectomized controls. The effects on pulsatile LH secretion of food reduction by 50% with or without dilution by cellulose to maintain food volume in orchidectomized rats were also examined. Food volume influences pulsatile LH secretion independent of macronutrient effect after 3 days of food restriction, but subsequently macronutrient deprivation predominates. The exaggerated increase in LH levels in orchidectomized rats subject to food restriction for 7 days was not due to immunochemical or chromatographic heterogeneity or alteration in biopotency of circulating LH molecules. Intravenously injected 125I-labeled rat LH analyzed by noncompartmental modeling revealed that neither LH clearance nor mean residence time was reduced by food restriction. We conclude that during food restriction in orchidectomized rats, increases in LH pulse amplitude exceed and precede the decreases in LH pulse frequency, although the early changes in pulse amplitude are predominantly due to reduced food volume rather than macronutrient deprivation.

Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C-II, and C-III.

ApoA-I and apoC-II are eluted in two isoforms and apoC-III2 is eluted in three isoforms by reversed phase high performance liquid chromatography (HPLC). The structural basis of these nongenetic heterogeneities was unravelled using HPLC of proteolytic peptides and time-of-flight secondary ion mass spectrometry (TOF-SIMS). In apoA-I, the chromatographic microheterogeneity was caused by the formation of methionine sulfoxides (MetSO). However, only residues Met112 and Met148 were found oxidized, whereas Met86 was unaffected and also resistant towards artificial oxidation. To assess whether and to what extent amino acid substitutions in apoA-I might affect methionine sulfoxidation, the tryptic peptides of 13 different mutant apoA-I proteins from 24 heterozygous apoA-I variant carriers were analyzed by HPLC. In normal apoA-I, the ratios MetSO112/Met112 and MetSO148/Met148 were highly variable. By contrast, the relative ratio of oxidation of methionine residues 112 and 148 was constant. The amino acid changes Lys107----Met, Lys107----O, Glu139----Gly, Glu147----Val, and Pro165----Arg resulted in the preferential oxidation of Met112, and Asp103----Asn resulted in a preferential oxidation of Met148; whereas Pro3----Arg, Pro3----His, Pro4----Arg, Asp89----Glu, Ala158----Asp, Glu198----Lys, and Asp213----Gly had no impact. ApoC-II and apoC-III isoforms differed by the oxidation of the two methionine residues in these proteins. Whereas in apoC-II both methionine residues were oxidized in parallel, in apoC-III the two methionine residues differed in their susceptibility towards oxidation. We conclude that the formation of MetSO depends on the molecular microenvironment within a protein.

High Resolution High Performance Liquid Chromatography Fingerprinting of Purified Human Chorionic Gonadotropin Demonstrates that Oxidation Is a Cause of Hormone Heterogeneity*

To characterize the structures of the various forms of hCG and its immunologically related fragments measured in blood, urine, and tissue culture media, we developed a highly sensitive HPLC tryptic fingerprinting system that can characterize as little as 15 micrograms of material. Standard preparations of the subunits of hCG purified from pregnancy urine were found to exhibit heterogeneity on reverse phase HPLC chromatography, complicating efforts to characterize such forms of hCG. The same type of chromatographic heterogeneity was observed when the hormone was reduced and S-carboxymethylated (RCM) and its RCM subunits separated on HPLC. One major and one minor peak were observed in the alpha- and beta-subunits from both native and RCM hormone, with a ratio of between 5:1 and 10:1. Development and application of a high resolution, semimicrobore HPLC fingerprint technique used together with an examination of selected peptides by amino acid sequencing and mass spectrometry defined the differences between these forms as oxidation. Using each of the isolated RCM subunit's major and minor HPLC peaks, the oxidation was shown to be reversible and probably due to interconversion of methionine with its sulfoxide form.

The chromatographic heterogeneity of rat transferrin on immobilized concanavalin A and lentil lectin.

A procedure was developed for the isolation of the microheterogeneous forms of rat transferrin consisting of anion-exchange and serial lectin affinity chromatographies. By deploying this technique, four to five different anionic species of the protein were detected in plasma. The two major components obtained, which encompassed 92-94% of the plasma transferrin, were further studied by sequential lectin chromatography. The larger of the two, representing 60-63% of plasma transferrin, was bound by concanavalin A - Sepharose, while the smaller one (30-32% of plasma transferrin) resolved into an unbound (25-27% of plasma transferrin) and a retarded (4-5% of plasma transferrin) fraction. The latter eluted from the column in a volume which was 1.9 times larger than that required for the passage of nonretarded transferrin. In accordance with their fucose contents, each of these three concanavalin A fractions resolved into a bound (20-29%) and an unbound (71-80%) subfraction by chromatography on lentil-Sepharose. It is concluded that there exist two kinds of glycan microheterogeneity in rat transferrin and that they are unrelated to each other. Consequently, at least six different forms of rat transferrin are available with respect to glycosylation. Epididymal fucosidase cleaved fucose from apotransferrin slowly and from the tryptic glycopeptide rapidly. Exploratory studies performed in vivo failed thus far to identify the significance of fucose in rat transferrin.

Development of anti-human colonic mucin monoclonal antibodies. Characterization of multiple colonic mucin species.

Structural relationships between colonic mucin species were assessed using a library of monoclonal antibodies (MAbs) directed against purified human colonic mucin (HCM). After immunization of mice with purified mucin from normal human colonic mucosa, 14% of 1,920 fusion products screened were positive for anti-HCM activity in a solid-phase assay. Patterns of selective binding by hybridomas to six discrete HCM species (I-VI) separated by DEAE-cellulose chromatography suggested the presence of both shared and species-specific antigenic determinants among HCM species I-VI. 23 anti-HCMs MAbs (7 IgM, 7 IgG1, and 9 IgG2) demonstrating a range of anti-HCM species specificities, were produced and used to study structural relationships between mucin species. Binding of various mucin species by individual anti-HCM MAbs was shown by competitive solid-phase radioimmunoassay to reflect the presence of identical epitopes on the different species. Adsorption of HCM species on a variety of affinity resins prepared with anti-HCM MAbs demonstrated that binding to multiple mucin species by a single MAb was related to intrinsic structural determinants. Four anti-HCM MAbs recognized protease-sensitive antigenic structures, which suggests that they may be directed to core HCM proteins. 12 of the anti-HCM MAbs were shown by solid-phase assay to recognize either complete (n = 5) or partial (n = 7) isolated colonic mucin oligosaccharide side chains of defined structure. Collectively, these data show the presence of both shared and unique antigenic structural determinants among colonic mucin species. Chromatographic heterogeneity of mucin glycoproteins seems to be related to the existence of biologically significant subclasses in the normal human colon.

Reversed-phase high-performance liquid chromatographic purification of subunits of oligomeric membrane proteins : The nuclear coded subunits of yeast cytochrome c oxidase

Reversed-phase chromatography of the subunits of an oligomeric membrane protein such as yeast cytochrome c oxidase requires additional sample handling techniques which are not necessary for soluble proteins. This paper considers these and discusses (1) methods for the removal of ballast material by preliminary batchwise extraction with solvent mixtures similar to those used for reversed-phase elution; (2) the chromatographic heterogeneity induced by partial cysteine oxidation; (3) the removal of tightly bound proteins from the stationary phase; and (4) the generation of an elution system with continuously variable selectivity based on acetonitrile-l-propanol ratios (0.05 % triethylamine, 0.05 % trifluoroacetic acid).These methods are designed to simplify complex mixtures of hydrophobic proteins prior to chromatography and to purify them chromatographically in high yield.

Structural and chromatographic heterogeneity of normal plasma fibrinogen associated with the presence of three γ-chain types with distinct molecular weights

Three different γ chains have been identified in fibrinogen isolated form normal human plasma with apparent molecular weights of 50000 (γ 50), 55000 (γ 55) and 57500 (γ 57.5), as shown by SDS-polyacrylamide gel electrophoresis. Plasma fibrinogen was separated by ion-exchange chromatography on DEAE-Sephacel into three populations of molecules, each differing in γ-chain composition. The first peak contained 87% of the total fibrinogen and was composed of molecules containing only γ 50 chains; the second peak included 3% of the fibrinogen and contained one γ 50 and one γ 55.5 chain and the third peak had 10% of the total which containedone γ 50 and one γ 57.5 chain, Cross-linked fibrin obtained from fibrinogen with only γ 50 chains contained γγ dimers exclusively of M r 100000, representing a uniform γ 50γ 50 dimer composition. The γγ dimers from purified fibrinogen of γ 50γ 55 type had molecular weights of 111000, 105000 and 100000, while dimers from γ 50γ 57.5 fibrinogen were M r 115000, 108000 and 100000. The relative proportions of γγ dimers from each purified fibrinogen population were consistent with random crosslinking of the γ monomers. The γ-chain identity of the variants was established by their conversion to covalent dimeric species after clotting by thrombin in the presence of Factor XIII, their incorporation of the fluorescent lysine analog dansyl cadaverine, by the staining intensity of monomeric and dimeric forms with the periodic acid-Schiff reagent after electrophoretic separation, and by plasmic degradation of all monomeric and dimeric forms in a pattern that was characteristic for γ chains. Individual γγ dimers were purified by preparative gel electrophoresis, and tyrosine was demonstrated to be the amino-terminal residue of all three γ chain types. We conclude that normal human fibrinogen can be separated into three populations of molecules due to molecular weight heterogeneity of their γ chains.

Fractionation of mouse alpha-fetoprotein.

4 distinct alpha-fetoprotein (AFP) containing fractions were obtained upon ion-exchange chromatography of late-gestational fetal mouse extracts. Despite this chromatographic heterogeneity, the individual AFP isolates were antigenically indistinguishable.

Insulin receptors in transformed fibroblasts and in adipocytes: a comparative study

This study compares the receptor for insulin in rat adipocytes with an insulin receptor from transformed AL/N mouse embryo fibroblasts (SVS AL/N). In many respects the receptors from the two tissue sources are very similar. Crude solubilized receptor preparations from both fat cells and SVS AL/N cells exhibit complicated insulin-binding kinetics (two binding plateaus) as well as an apparent chromatographic heterogeneity (Sepharose 6B); each receptor preparation yields two insulin-binding components having apparent Stokes radii of about 7.2 and 3.8 nm. In contrast, the receptors isolated from both cell types by affinity chromatography behave as single chromatographic components (Sepharose 6B) having apparent stokes radii of 3.8 nm and exhibit simple insulin-binding kinetics without evidence for receptor–ligand co-operativity. The isolated receptor from SVS AL/N cells (KD about 1.2 nM) possesses a slightly lower insulin affinity than does the receptor from fat cells (KD about 0.8 nM); nonetheless, the SVS AL/N receptor exhibits an appropriate ligand specificity for insulin and insulin analogues. Receptors from both cell types are similarly sensitive to trypsin and exhibit an identical dependence on pH for insulin binding. Differences in the two receptor preparations are observed in the effect of NaCl and phospholipase C, reagents which augment insulin binding in adipocyte membranes but do not affect the binding by SVS AL/N cell membranes. Additionally, iodoacetamide, N-ethylmaleimide, and ethanol markedly reduce the binding by SVS AL/N membranes, whereas the binding by adipocyte membranes is unaffected. It is concluded that certain differences between the receptors from the two sources may be attributed to differences in the membrane environment in which the receptors reside. However, in view of the many similarities between the two receptor preparations, it is suggested that the same receptor macromolecule may participate in the response of a variety of cell types not only to insulin but also to other insulin-like polypeptides.

Glycosylation of interferons. Effects of tunicamycin on human immune interferon

Human immune interferon, induced in leukocytes by phytohemagglutinin, was prepared in the absence and presence of tunicamycin, an antibiotic which selectively inhibits the glycosylation of newly synthesized glycoproteins. Interferon preparations, produced in the absence of the antibiotic, displayed a considerable chromatographic heterogeneity on: (a) concanavalin A-agarose, (b) phenyl-agarose, (c) Cibacron Blue F3GA-agarose, and (d) polyuridylic acid-agarose. This heterogeneity was completely eliminated when tunicamycin (2 microgram/ml) was present during induction of interferon; all activity was then recovered in the breakthrough fractions from all sorbents. The level of interferon activity in leukocyte culture fluid was not affected by tunicamycin within the range of concentration 0.05 to 2.0 microgram/ml. These data indicate that (a) human immune interferon undergoes glycosylation, and tunicamycin is an effective inhibitor of this process. Thus, it appears that (b) at least some of the carbohydrates of human immune interferon are N-glycosidically linked. Moreover, it seems that (c) glycosylation is not necessary for an interferon molecule to either be secreted by the cell or (d) to express its antiviral function. Such properties of human immune interferon as (e) the apparent hydrophobicity and (f) an affinity for a polyribonucleotide are conferred only when its glycosylation is unimpaired.

Characteristics of the solubilized insulin receptor of human placenta

A soluble human insulin receptor can be obtained in high yield from placenta membranes, using the detergent Ammonyx-LO. In crude soluble preparations, the placenta receptor exhibits complex insulin binding kinetics (two binding plateaus, half-saturated at about 40 pM and 700 pM insulin) and an apparent chromatographic heterogeneity (Sepharose 6B) with two insulin binding components having apparent Stokes radii of 72Å and 38Å. However, subsequent to purification by affinity chromatography on insulin-Sepharose, the placenta receptor exhibits a simple insulin binding isotherm, without evidence for binding cooperativity (K D about 830 pM), and upon chromatography behaves as a single component with a Stokes radius of 38Å A. When combined with a previously described non-receptor glyco-protein preparation isolated from liver membranes, the affinity-purified plancenta receptor undergoes an increase in its Stokes radius from 38Å to 72Å. Because of the physicochemical similarities between the placenta receptor and the insulin receptor previously isolated from liver cell membranes, it is concluded that the placenta receptor is representative of the insulin receptor present in other traditional target tissues for insulin. The study underscores a possible role for insulin in placental physiology and provides for the large scale isolation of the human receptor from a readily available source.

Insulin receptor: Interaction with nonreceptor glycoprotein from liver cell membranes

In crude receptor preparations (either particulate or soluble) of rat liver membranes, the insulin receptor exhibits complicated binding kinetics (two binding plateaus, half-saturated at approximately 60 pM and 700 pM insulin) and an apparent chromatographic heterogeneity, suggested by the presence of two detectable, soluble insulin-binding components with apparent Stokes radii of 72 A and 38 A. In contrast, the insulin receptor isolated by affinity chromatography exhibits a simple binding isotherm (half-maximal saturation of binding at 700 pM insulin) without evidence for negative cooperativity and behaves as a single component (apparent Stokes radius of 38 A) upon chromatography on Sepharose 6B. The apparent discrepancies between the properties of the unpurified insulin receptor and the affinity-purified receptor can be attributed to the presence in crude preparations of a nonreceptor constituent(s) having properties consistent with those of a membrane glycoprotein. A glycoprotein fraction from such crude soluble membrane preparations, freed from insulin receptor and subsequently partially purified using concanavalin-A-agarose, when combined with affinity-purified insulin receptor, causes both a reappearance of the complicated binding kinetics and an increase in the receptor's apparent Stokes radius from 38 A to 72 A. Similar results are observed for a glycoprotein fraction obtained from rat adipocyte membranes but are not observed for an identical fraction isolated from human erythrocyte membranes. We conclude that the insulin receptor in rat liver membranes can interact with another nonreceptor membrane glycoprotein that may represent either a nonrecognition moiety of the receptor oligomer or an effector molecule to the biological action of insulin.

Chromatographic Heterogeneity of Insulin Extracted from Insulomas

On gel filtration of acid-ethanol extracts from three pancreatic beta-cell adenomas 1.4% to 1.8% of total immunomeasurable insulin (IMI) eluted ahead of proinsulin. This high molecular IMI was resolved into three components. The presence of urea in the dilute acetic acid solutions of extracted tumor tissue did not influence the pattern of gel filtration. High molecular IMI dissolved in dilute acetic acid showed to be stable if immediately rechromatographed, but a partial dissociation to insulinlike and proinsulinlike components (ILC and PLC) was found if rechromatography was performed after 48 h of incubation. Mainly ILC and PLC were found on rechromatography provided high molecular IMI was dissolved and incubated briefly in 0.04M phosphate buffer, pH 7.4. It proved improbable that the proteolytic action of some protein being extracted with the hormones caused a splitting of high molecular IMI at pH 7.4. We conclude from our findings that the components of high molecular IMI are not precursors of proinsulin and insulin but are either self-associated products of the hormones or associations of insulin and proinsulin to other proteins extracted from insuloma tissue.

Chromatographic heterogeneity of 3-hydroxyproline and its basis

trans-3-Hydroxy- l-proline is frequently eluted from amino acid analyzer columns as a double or asymmetric peak. This behavior was shown to depend on the size of the hydroxyproline sample, the pH, and the nature of the eluting buffer. Comparison of a number of organic acids used as eluting buffers suggested that the hydroxyl group of malic or citric acid participates in reversible complex formation with 3-hydroxyproline. Evidence for such chemical interaction was obtained by NMR spectrometry.

Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides.

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and beta-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

Multiple Forms of Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase in Xenopus laevis: LEVELS OF ACTIVITY DURING OOCYTE AND EMBRYONIC DEVELOPMENT

Abstract The amounts of the various forms of RNA polymerase (Ia, Ib, IIa, IIb, and III) have been determined in oocytes, eggs, and embryos of Xenopus laevis. During oogenesis the relative proportions of the enzymes remain nearly constant, although the absolute levels of all forms increase dramatically. As a result, the mature oocyte, as well as the unfertilized egg, contains 4 to 5 orders of magnitude more RNA polymerase activity than is present in an individual somatic cell. Both oocytes and eggs contain unusually high levels of forms III and IIa in addition to forms I and IIb which predominate in somatic cells. The amounts and relative proportions of the five enzymes in the unfertilized egg remain about the same up to gastrulation of the embryo. During subsequent stages of embryogenesis the total amounts of forms I and IIb increase about 10-fold while the amounts of forms IIa and III increase only slightly. The rapid increase in cell number along with this selective increase in forms I and IIb during early development re-establishes the same relative and absolute enzyme levels per cell in the swimming embryo that are present in adult somatic cells. Anucleolate mutant embryos at the early swimming stage have the same amount of each enzyme as do control embryos. Furthermore the relative amounts of the various enzymes in purified nuclei are the same in anucleolate embryos as they are in control embryos. Chromatographic heterogeneity in enzyme I was found at all stages of oocyte maturation and throughout embryogenesis of control and anucleolate mutant embryos.

Comparison of methyl-labeled tRNA in a neoplastic cell line with a paired non-neoplastic control

A comparison has been made between methylation of tRNA in intact cells from a non-neoplastic mouse cell line and that from two related neoplastic mouse cell lines. By adding 3H-methyl-methionine and 14C-methyl-methionine to the growth medium, it was possible to separately label and then to simultaneously extract, isolate, and chromatograph the tRNA from neoplastic and non-neoplastic cells. Isotope ratios were calculated from reversed-phase chromatography profiles and a consistently reproducible variation in the ratio was seen through the region where the first tRNA's were eleuted, indicating chromatographic heterogeneity. Although this difference between tRNA methylation in non-neoplastic and that in neoplastic cells is small, the data are consistent with the view of Srinivasan and Borek that aberrant methylation may be a factor in neoplastic conversion.

Correlation of Charge Properties of Human Reaginic Antibodies with Charge of the Corresponding Allergens

Abstract Reaginic (skin-sensitizing) antibodies in the sera of ragweed allergic individuals show considerable heterogeneity in elution properties when chromatographed on the anion exchanger, DEAE-Sephadex (1, 2). Evidence was obtained (3) that the chromatographic heterogeneity was primarily due to charge and not to size differences between the reaginic antibody molecules. A basis for charge heterogeneity of antibody molecules in general was suggested by the recent finding of Sela and Mozes (4) that specific rabbit antibodies vary in their overall charge depending (inversely) on the net charge of the corresponding immunogen. The availability of two purified ragweed pollen allergens, antigen Ra.3 (5) and antigen E (6), which differ in net charge as well as other physicochemical and immunologic properties, made it possible to investigate whether a similar relationship might obtain for human reagins formed to the allergenic constituents of ragweed pollen.

Electrophoretic Separation of Bovine Sarcoplasmic Proteins Fractionated by DEAE-Cellulose Chromatography

Heterogeneity of bovine sarcoplasmic protein fractions obtained by DEAE-cellulose ion exchange chromatography was investigated by vertical polyacrylamide gel electrophoresis. Results of these electrophoretic analyses indicated that the five major chromatographic fractions were quite heterogeneous. Fraction areas I and III, which appeared as single chromatographic peaks, showed five and six distinct bands, respectively, when subjected to gel electrophoresis. The other chromatographic groups, fraction areas II, IV and V, were separated into five, eight and three electrophoretically different bands, respectively. Some possible reasons for the apparent chromatographic heterogeneity are discussed.