High-performance Gel Chromatography Research Articles

Characterization of further association of the trimeric membrane protein porin by low-angle laser-light scattering photometry coupled with high-performance gel chromatography

Porin (OmpF), a trimeric membrane protein, in an extract of the outer membrane of Escherichia coli gave a twin-peaked elution pattern on Sephacryl S-300HR gel chromatography in the presence of sodium dodecyl sulphate. The species eluting earlier and later were found to be the hexamer and trimer, respectively, from molecular mass determination by low-angle laser-light scattering photometry coupled with TSK-G3000SWXL gel chromatography. As the hexamer was dissociated into the trimer under the conditions of sodium dodecyl sulphate polyacrylamide gel electrophoresis, its presence had been overlooked. The addition of lipopolysaccharide, another component of the outer membrane, and subsequent dialysis induced association of the trimer, the product containing an appreciable amount of the hexamer.

Cross-Linking of Egg White Riboflavin-Binding Protein by Calcium Phosphate

No larger molecular weight component appeared in the high-performance gel chromatogram when calcium alone was added to the riboflavin-binding protein solution (RfBP), indicating that calcium alone did not aggregate it. RfBP bound calcium, but the amount of bound calcium decreased markedly upon dephosphorylation. Cross-linked RfBP, which was detected by high-performance gel chromatography using simulated milk ultrafiltrate as the effluent, was formed when calcium and phosphate were added. Cross-linking of RfBP was confirmed by ultracentrifugal analysis. RfBP was found to be cross-linked through its phosphate groups, since no cross-linked fraction was detected when calcium and phosphate were added to a dephosphorylated RFBP solution. RfBP cross-linked by calcium phosphate formed at 12-20 mM calcium, 13-17 mM phosphate, and 10 mM citrate was a dimer, since its retention time was consistent with that of the dimer cross-linked by glyceraldehyde. The physiological function of phosphate groups of RfBP was also discussed.

High performance gel and ion-exchange chromatography of buffalo casein

Buffalo casein was separated into 3 fractions by high performance gel chromatography on a TSK-GEL G3000SW column using 50 mM phosphate buffer (pH 6·7) containing 6 m urea and 0·3 m NaCl. The first and second eluted fractions contained κ- and α s 2 -caseins, respectively. Buffalo casein was also separated into 5 fractions (κ-, unidentified, β-, α s 2 - and α s 1 -caseins) by high performance ion-exchange chromatography on a TSK-GEL DEAE-5PW column, although the α s 1 - and α s 2 -casein fractions overlapped. The relative composition of Egyptian buffalo casein was 36·9% α s 1 -casein, 9·3% α s 2 -casein, 35·8% β-casein, 13·2% κ-casein and 4·8% unidentified casein when κ- and α s 2 -casein contents were estimated by high performance gel chromatography and the other caseins by high performance ion-exchange chromatography.

Polydisperse Dextran as a Diffusing Test Solute to Study the Membrane Permeability of Alginate Polylysine Microcapsules

Applications of alginate polylysine (APL) microcapsules in cell culture engineering and hybrid artificial organs require strict control of membrane permeability. For example, hybrid artificial organs must permit the diffusion of smaller molecules including peptides and proteins and the exclusion of leucocytes and immunoglobulins (MW > 150,000). Single molecular weight solutes such as proteins have been used to study the molecular weight cut-off of the membrane. A new approach that uses a heterogenous mixture of dextrans of different molecular weights: -MW 10,000-500,000 as a test solute in diffusion experiments is described. Intra/extra capsular changes in concentration and molecular weight distribution of the heterogenous dextran mixture are monitored and determined by high performance gel chromatography. MW can then be related to diffusional Stokes radius, a more useful parameter when comparing permeability of cell membrane to different test solutes.

Polymerization of casein on heating milk

The content of polymerized casein formed on heating casein micelles was determined by high-performance gel chromatography on a TSK-GEL G3000SW column after dissociating casein micelles with 6 m urea, EDTA and 2-mercaptoethanol. Polymerized casein was detected in whey protein-free (WPF) milk, prepared by dispersing casein micelles in milk ultrafiltrate, heated at 80°C for 45–75 s, and increased markedly above 120°C. Lactose was considered to be the main constituent in milk affecting the polymerization of casein on heating since the content of the polymerized casein was lower in casein micelle dispersions containing no lactose than in WPF milk and the polymerized casein increased when lactose was added to casein micelle dispersion. The amount of polymerized casein formed on heating decreased when casein micelles were disaggregated into submicelles, suggesting that the aggregation of casein, such as in micelles, promotes the polymerization of casein on heating.

Improvement of the Surface Functional Properties ofβ-Lactoglobulin andα-Lactalbumin by Heating in a Dry State

Controlled heating in a dry state greatly improved the surface functional properties of whey proteins (β-lactoglobulin and α-lactalbumin). Although whey proteins were completely insolubilized by heating at 80°C in an aqueous solution, their solubility was kept even after heating at 80°C in a dry state (7.5% moisture content) for 5 days. The surface hydrophobicity of α-lactalbumin was increased during the dry-heating, while that of β-lactoglobulin was decreased. In addition, the fluorescence spectra excited at 280 nm of dry-heated whey proteins suggested the significant conformational changes. High-performance gel chromatography showed that a considerable amount of soluble aggregates was formed in the dry-heated β-lactoglobulin, while a small amount of soluble aggregate was observed in the dry-heated α-lactalbumin. The foaming properties of dry-heated whey proteins were increased to about 3 times that of untreated proteins. The emulsifying properties of dry-heated whey proteins were also increased, compared to untreated proteins, although a slight decrease in the emulsion stability was observed in dry-heated β-lactoglobulin. The improvement of the surface properties seemed to come from the partial unfolding suitable for the formation of foam film and the entrapment of oil droplets.

Molecular weight determination and compositional analysis of dextran-protein conjugates using low-angle laser light scattering technique combined with high-performance gel chromatography

The low-angle laser light scattering technique combined with high-performance gel chromatography was applied for characterization of the dextran-ovalbumin and dextran-lysozyme conjugates obtained from the mild heating in dry state, which is attracting interest as a way leading to stabilization of proteins and to production of proteins with excellent emulsifying or antimicrobial ability (Nakamura, S., Kato, A. and Kobayashi K. (1991) J. Agric. Food Chem. 39, 647–650). According to the above technique, providing the information about the molecular weight distribution and the composition of the conjugates, one or two dextran molecules were found to be linked to one molecule of the proteins. In addition, each of the conjugates was shown to exist as an oligometric assembly of which formation is promoted by an increase in the salt concentration of buffer. The observations suggest that the increase in the hydrophobicity of the protein moiety as a result of partial denaturation and the introduction of the hydrophilic dextran chain affords the conjugate an amphiphilic property.

Purification and characterization of a novel sialidase found in procyclic culture forms of Trypanosoma brucei

A membrane-bound sialidase (EC 3.2.1.18) was found in procyclic trypomastigotes of Trypanosoma brucei. The mammalian stage bloodstream form, however, displayed no sialidase activity. This sialidase is an integral surface protein, linked to the membrane via a glycosylphosphatidylinositol anchor. After osmotic lysis and solubilization with Triton CF-54, the enzyme was purified 1900-fold by gel filtration and ion exchange chromatography. Its size, as determined by conventional and high-performance liquid gel chromatography, is 67 kDa. The sialidase is active over a broad pH and temperature range with optima at pH 6.9 and 35°C, respectively. No loss of activity is observed after 4 freeze-thaw cycles. T. brucei sialidase activity is inhibited by N-(4-nitrophenyl)oxamic acid and 2-deoxy-2,3-didehydro- N-acetylneuraminic acid, the latter, however, being less effective. N-Acetylneuraminic acid shows no inhibitory effect, whereas a variety of metal ions are potent inhibitors. The sialidase is activated by di- and tricarboxylic acids, but inhibited by chloride. Relative hydrolysis rates of various sialic acid-containing compounds reveal that de- O-acetylated bovine submandibular gland mucin is the preferred substrate and that α(2–3)-linkages are hydrolyzed faster than α(2–6)-linkages.

Analytical and micropreparative high-performance gel chromatography of proteins with a short column: Determination of molecular size, rapid determination of ligand binding constant and purification of S-carboxymethylated proteins for microsequencing

A short (75-mm) column of TSK-GEL G3000SW (S-column) can be used for both analytical and micropreparative gel chromatography. The elution conditions for the S-column were optimized and thereafter the column was well calibrated under native and denaturing conditions in a manner similar to that with a 600-mm column. The error in the Stokes radii of proteins obtained with the S-column was treated statistically. This S-column has the advantage of the economy in the time taken for analysis and solvent exchange and in the sample amounts required. Hence, it is useful for studying protein—ligand interactions by Hummel—Dreyer gel chromatography and for purifying S-carboxymethylated proteins prepared for microsequencing. These were exemplified by the analysis of the binding of o-nitrophenol to catechol 2,3-dioxygenase and of the NH 2-terminal amino acid sequence of rat pancreatic prophospholipase A 2.

The Least Number of Phosphate Groups for Crosslinking of Casein by Colloidal Calcium Phosphate

Artificial casein micelles were formed with whole human casein at 20mM calcium, 17mM phosphate, and 10mM citrate. The casein micelles disaggregated by 6 M urea were separated by high performance gel chromatography on a TSK-GEL G4000SW column into crosslinked and monomeric fractions. When the crosslinked casein fraction was analyzed by high performance ion-exchange chromatography on a TSK-GEL DEAE-5PW column, a small peak, representing the 3-P component of human β-casein, and distinct peaks of the 4-P and 5-P components were found. Artificial casein micelles were formed from mixtures of each purified component of human β-casein and bovine κ-casein, disaggregated by urea, and separated on a TSK-GEL G4000SW column. The casein aggregates crosslinked by colloidal calcium phosphate were formed in artificial casein micelles of the 3-P and 4-P components. In contrast, no crosslinkage was formed in artificial micelles of the 1-P and 2-P components. The results indicate that at least three phosphate groups are needed for crosslinking of casein by colloidal calcium phosphate.

Analysis of polymer molecular weight distributions in aqueous two-phase systems

The partitioning of proteins and other biomaterials between two aqueous phases containing polyethyleneglycol and dextran is a strong function of the molecular weight of the two polymers. Although both polymers are polydispersed (especially Dx) most theoretical treatments refer only to the average molecular weight (number or mass) and assume that the molecular weight distribution of each polymer is the same in both phases. In this work the molecular weight distributions of four stock solutions of PEG (4000, 6000, 10,000 and 20,000) and four stock solutions of Dx (10,000, 40,000, 110,000 and 500,000) were measured using High Performance Gel Chromatography. The measurements were repeated on the phases formed by the polymer solutions after they were mixed and allowed to equilibrate. The molecular weight distribution of the Dx differed in the top and bottom phase; both differed from that of the stock solution. Although we believe that the molecular weight distribution for PEG also differs in the top and bottom phases, we were unable to determine this within the resolution of our instruments.

Influence of total lipid concentration, bile salt:lecithin ratio, and cholesterol content on inter-mixed micellar/vesicular (non-lecithin-associated) bile salt concentrations in model bile.

We modified classic equilibrium dialysis methodology to correct for dialysant dilution and Donnan effects, and have systematically studied how variations in total lipid concentration, bile salt (taurocholate):lecithin (egg yolk) ratio, and cholesterol content influence inter-mixed micellar/vesicular (non-lecithin-associated) concentrations (IMC) of bile salts (BS) in model bile. To simulate large volumes of dialysant, the total volume (1 ml) of model bile was exchanged nine times during dialysis. When equilibrium was reached, dialysate BS concentrations plateaued, and initial and final BS concentrations in the dialysant were identical. After corrections for Donnan effects, IMC values were appreciably lower than final dialysate BS concentrations. Quasielastic light scattering was used to validate these IMC values by demonstrating that lipid particle sizes and mean scattered light intensities did not vary when model biles were diluted with aqueous BS solutions of the appropriate IMC. Micelles and vesicles were separated from cholesterol-supersaturated model bile, utilizing high performance gel chromatography with an eluant containing the IMC. Upon rechromatography of micelles and vesicles using an identical IMC, there was no net transfer of lipid between micelles and vesicles. To simulate dilution during gel filtration, model biles were diluted with 10 mM Na cholate, the prevailing literature eluant, resulting in net transfer of lipid between micelles and vesicles, the direction of which depended upon total lipid concentration and BS/lecithin ratio. Using the present methodology, we demonstrated that inter-mixed micellar/vesicular concentrations (IMC) values increased strongly (5 to 40 mM) with increases in both bile salt (BS):lecithin ratio and total lipid concentration, whereas variations in cholesterol content had no appreciable effects. For model biles with typical physiological biliary lipid compositions, IMC values exceeded the critical micellar concentration of the pure BS, implying that in cholesterol-supersaturated biles, simple BS micelles coexist with mixed BS/lecithin/cholesterol micelles and cholesterol/lecithin vesicles. We believe that this methodology allows the systematic evaluation of IMC values, with the ultimate aim of accurately separating micellar, vesicular, and potential other cholesterol-carrying particles from native bile.

Molecular Weight Determination of Phospholamban Oligomer in the Presence of Sodium Dodecyl Sulfate: Application of Low-Angle Laser Light Scattering Photometry1

The number of polypeptides constituting the oligomeric structure of canine phospholamban (a putative regulator of Ca(2+)-ATPase of cardiac sarcoplasmic reticulum) stable even in the presence of sodium dodecyl sulfate was estimated through determination of the molecular weight of the oligomer. Owing to the small molecular size, the low UV-absorptivity and the limited availability, the molecular weight determination required very sophisticated application of the following technique, used as the only recourse: low-angle laser light scattering measurement combined with high-performance gel chromatography. The molecular weight of phospholamban oligomer was found to be 30,400 and the number of subunits was concluded to be five after correction for the dependence of the apparent molecular weights on the protein concentration.

Rapid on-line determination of cholesterol distribution among plasma lipoproteins after high-performance gel filtration chromatography

A high-performance gel chromatography (HPGC) system has been developed which allows the unattended on-line determination of lipoprotein cholesterol distribution (VLDL-C, LDL-C, HDL-C), within 40 min, in microliter quantities of plasma using a single, relatively inexpensive column (Superose 6HR). The FAST cholesterol reagent (Sclavo) and a knitted PFTE Kratos reaction coil (Applied Biosystems) were found to provide optimal sensitivity, linearity, resolution, and dispersion characteristics. Validation is provided by comparison to target values for human quality control reference sera, and by comparing the values obtained by HPGC to the beta-quant method (LRC). The utility of the system is illustrated by comparing profiles from seven different species with normal or elevated plasma cholesterol concentrations. This technique allows rapid analysis of samples, regardless of species, without the use of precipitating agents or the ultracentrifuge. It could also be applied for the direct clinical determination of LDL-cholesterol.

Protein-protein interaction of detergent-solubilized Ca 2+-ATPase during ATP hydrolysis analyzed by low-angel laser light scattering photometry coupled with high-performance gel chromatography

Protein—protein interaction of detergent-solubilized Ca 2+-ATPase was examined, employing low-angle light scattering photometry coupled with high-performance gel chromatography. When solubilized with octa(ethylene glycol) mono- n-dodecyl ether (C 12E 8) and chromatographed in the presence of 0.3 mg/ml C 12E 8, the Ca +-ATPase emerged as a single peak with an intermediate molecular weight between the monomer and the dimer, showing a dissociation-association equilibrium of the two components. In the presence of 50 μg/ml phosphatidylcholine and 0.3 mg/ml C 12E 8 at 0°C, the Ca 2+-ATPase (0.8 mg) emerged as the two distinct components with molecular weights of 125 000±2100 ( n = 3) and 211 300±7300 ( n = 3), indicating that there was no rapid interconversion between the monomer and the dimer. Under the latter conditions, addition of ATP induced fusion of the two components. The apparent molecular weight of the fused peak shifted from the monomer to the dimer as the amount of protein increased. Addition of ADP or adenosine 5′-(β, γ-methylene triphosphate), however, did not induce such fusion of the peaks.The ATP-induced fusion of the peaks was not observed either in 5 mM CaCl 2, the conditions in which the rate of ATP hydrolysis was extremely slow. Thus, the solubilized Ca 2+-ATPase underwent a rapid interconversion between the monomer and the dimer during ATP hydrolysis. These results suggest that the protein-protein interaction during ATP hydrolysis is an intrinsic nature of Ca 2+-ATPase and that such interaction may be important for Ca 2+ transport by Ca 2+-ATPase in the sarcoplasmic reticulum membranes.

Cleavage of the linkage between colloidal calcium phosphate and casein on heating milk at high temperature

SummaryIn order to examine the effect of heating on the changes in the linkage between colloidal Ca phosphate (CCP) and casein, high-performance gel chromatography of casein micelles disaggregated by 6 M-urea was carried out using 6 M-urea simulated milk ultrafiltrate as the effluent. Although the CCP content increased when whey protein-free (WPF) milk was heated at 60–90 °C for 10 min, almost no changes in the content of casein aggregates cross-linked by CCP were observed. The content of casein aggregates cross-linked by CCP decreased from 51·9 to 46·1% in WPF milk and from 52·3 to 43·6% in concentrated WPF milk on heating at 135–140 °C for 75 s, indicating the cleavage of the linkage between CCP and casein. The cleavage of the linkage between CCP and casein on heating was considered to occur without liberation of ester phosphate groups. It was suggested that the transformation of CCP to another form was responsible for the cleavage between CCP and casein on heating milk at high temperature

Measurement of molecular weight distribution pattern of organic substances dissolved in environmental waters by an automatic high-performance gel chromatography.

An apparatus was developed for automatic measurement and to evaluate the pattern of molecular weight distribution of organic substances dissolved in water. A separation column (7.6 mm i.d., 25 mm in length) packed with Asahipak GS-220 was used. Distilled water was used as the mobile phase at flow rate of 1 ml/min, and a chromatogram was obtained by a high sensitive refractive index detector. The apparatus was regulated automatically by a system-controller. Sample size necessary for an analysis was 0.5 ml of filtrated environmental water without preconcentration, and one chromatogram was obtained in twenty minutes. Some enviromental water gave different chromatographic patterns. River water was measured by the apparatus continuously and automatically and chromatograms were obtained once an hour, which showed the change of the patterns from morning to evening. The apparatus was found to be suitable for an automatic measurement of organic substances dissolved in enviromental waters.

Minimum enzyme unit for Na +/K +-ATPase is the αβ-protomer. Determination by low-angle laser light scattering photometry coupled with high-performance gel chromatography for substantially simultaneous measurement of ATPase activity and molecular weight

The oligomeric state of canine renal Na +/K +-ATPase solubilized by octaethylene glycol n-dodecyl ether (C 12E 8) was studied by means of low-angle laser light scattering photometry coupled with high-performance gel chromatography (HPGC). At around 0°C the solubilized enzyme was separated into the (αβ) 2-diprotomeric and αβ-protomeric protein components with M r values of 302 000 ± 10 000 and 156 000 ± 4000, respectively, in approximately equal quantities. As the temperature of chromatography was increased toward 20°C, the two protein components converged into a single major component. The M r of this component depended on the monovalent cation included in the elution buffer, and was 255 000 or 300 000 in the presence of 0.1 M NaCl or 0.1 M KCl, respectively. A computer simulation technique showed that the solubilized enzyme was in a dissociation-association equilibrium of 2 promoters ⇌ diprotomer at 20°C, and the difference in apparent M r of the solubilized enzyme between the two species of monovalent cation was interpreted by an association constant ( K a) in the presence of 0.1 M KCl that was about 50-fold larger than in the presence of 0.1 M NaCl. In order to measure ATPase activity and M r of the solubilized enzyme simultaneously, a TSKgel G3000SW column had been equilibrated and was eluted with an elution buffer containing 0.30 mg/ml C 12E 8 and 60 μg/ml phosphatidylserine (bovine brain) as well as the ligands necessary for the enzyme to exhibit the activity at pH 7.0 and 20°C. The solubilized enzyme was always eluted as a single protein component irrespective of the amount of the protein applied to the column, ranging between 240 and 10 μg. The M r of the protein component, however, decreased from 214 000 to 158 000 with the decrease of the protein amount. The specific ATPase activity, however, remained constant at a level of 64 ± 4% of that of the membrane-bound enzyme even in the range of protein concentration sufficiently low as to allow the enzyme to exist only in the protomeric form. Thus, the αβ-protomer is concluded to be the minimum functional unit for the ATPase activity. The value of K a obtained from the concentration-dependent dissociation curve was 5 · 10 5 M −1 for the enzyme turning over, and 1.1 · 10 7 M −1 for the enzyme inhibited with ouabain. It was discussed, based on the values of K a obtained, that the enzyme would exist as the diprotomer or the higher oligomer in the membrane.

High molecular weight insulin-like growth factor binding protein complex: Purification and properties of the acid-labile subunit from human serum

Abstract In the human circulation, the insulin-like growth factors (IGFs) circulate as part of a growth hormone-dependent 125- to 150-kDa complex. This complex has been postulated to contain, in addition to IGFs and one or more IGF-binding proteins, an acid-labile subunit (ALS) which does not itself bind IGFs. In this study, the ALS has been purified 1600-fold from human serum, and its binding properties have been examined. Fresh serum was fractionated on DEAE-Sephadex, and active fractions (determined by radioimmunoassay) were purified by affinity chromatography on an IGF-agarose column saturated with the plasma IGF-binding protein BP-53. After further high performance anion exchange chromatography, an ALS preparation was obtained which contained only an 84-86-kDa protein doublet, converting to a single 70-kDa band on N-glycanase treatment, and having an amino-terminal sequence unrelated to IGF-binding proteins or receptors. Pure ALS formed a complex with BP-53 (Ka approximately 5 x 10(8) M-1), immunoprecipitable by anti-BP-53 antiserum, only in the presence of IGF-I or IGF-II. This complex appeared at approximately 150 kDa on high performance gel chromatography. Pure ALS had no intrinsic IGF-binding activity and no effect on the binding of IGF-I or IGF-II to BP-53. These studies suggest that formation of the high molecular weight IGF-binding protein complex requires ALS, BP-53, and IGF.

Subunit components of casein micelles from bovine, ovine, caprine and equine milks

SummarySubunit components of ovine, caprine and equine casein micelles were separated by gel chromatography using a TSK-G4000SW high-performance column and the subunit components of the fractions analysed and compared with bovine casein. Molecular weights of the casein complexes were determined by the combined use of high-performance gel chromatography and low-angle laser light scattering. The caprine and ovine caseins were separated into three peaks (F2, F3 and F4) which were similar to those of bovine casein with respect to composition and molecular weight (500, 100 and 23 K). These F2, F3 and F4 peaks consisted mainly of αs- and κ-casein, αs- and β-casein and β-casein respectively. The equine casein was separated into two components corresponding to F3 and F4 of bovine casein. These F3 and F4 peaks consisted mainly of αs- and β-casein and β-casein respectively. The molecular weight of equine F3 (850 K) was different from that of the other three species. The contents of F2 and F4 in these caseins were dependent on the contents of κ-casein and β-casein respectively.