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

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

Rapid method for trehalulose production and its purification by preparative high-performance liquid chromatography

Trehalulose was produced with a good yield by enzymatic conversion of sucrose and easily purified by preparative HPLC using a single Ca2+-based column. In addition, the structure of this sugar was confirmed by 13C and 1H n.m.r studies.     

A preparative method for isolation of fucoxanthin from Eisenia bicyclis by centrifugal partition chromatography

Introduction – Eisenia bicyclis (Kjellman) Setchell (Laminariaceae) is a common brown alga that inhabits around the coast of Korea, Japan and China. It contains fucoxanthin, a major carotenoid of brown algae which shows a variety of pharmaceutical functions. Objective – The aim of this investigation was the quantification and preparative isolation of fucoxanthin from fresh E. bicyclis using a new separation scheme, centrifugal partition chromatography (CPC). Methodology – The fucoxanthin fraction (Fuco fraction) was prepared by solvent partition method from the acetone extract of fresh E. bicyclis. Fuco fraction was used for CPC using a two‐phase solvent system of n‐hexane–ethyl acetate–ethanol–water (5:5:7:3, v/v/v/v). The flow rate of mobile phase was 2 mL/min with descending mode while rotating at 1000 rpm. The eluate was monitored at 410 nm. The content and structure of fucoxanthin in the CPC fraction were confirmed with HPLC, UV, APCI/MS and NMR spectra. Results – A preparative CPC yielded 20 mg of fucoxanthin (87% recovery from Fuco fraction) in a two‐step separation from 516 mg of Fuco fraction containing 4.59% fucoxanthin. The purity of the isolated fucoxanthin was about 81% in the first CPC step and over 98% in the second CPC step based on the calibration curve. The isolated fucoxanthin was identified as all‐trans‐fucoxanthin with APCI/MS (parent ion at m/z 641 [M + H ? H₂O]⁺) and ¹H, ¹³C and 2‐D NMR spectra. Conclusion – High‐purity fucoxanthin was successfully isolated from fresh E. bicyclis, suggesting further potential applications in the industrial use of this valuable carotenoid. Copyright © 2011 John Wiley & Sons, Ltd.     

Biotin-binding protein from chicken egg yolk. Assay and relationship to egg-white avidin.

1. Biotin in chicken egg yolk is non-covalently bound to a specific protein that comprises 0.03% of the total yolk protein (0.8 mg/yolk). This biotin-binding protein is not detectable by the normal avidin assay owing to the biotin being tightly bound. Exchange of [14C]biotin for bound biotin at 65 degrees C is the basis of an assay for this protein. 2. Biotin-binding protein from egg yolk is distinguishable from egg-white avidin on Sephadex G-100 gel filtration, although the sizes of the two proteins appear quite similar. 3. Biotin-binding protein is denatured at a lower temperature and freely exchanges biotin at lower temperatures than does avidin. 4. The biotin-binding protein in egg yolk is postulated to be responsible for the deposition of biotin in egg yolk. D-[carboxyl-14C]Biotin injected into laying hens rapidly appears in the egg bound to yolk biotin-binding protein and avidin. Over 60% of the radioactivity is eventually deposited in eggs. The kinetics of biotin deposition in the egg suggests a 25 day half-life for an intracellular biotinyl-coenzyme pool in the laying hen.     

Isolation and characteristics of carbohydrate chains of riboflavin-binding glycoprotein from the chicken egg

Isolation and characterization of oligosaccharides of riboflavin binding glycoprotein from hen white is described. Reductive cleavage of the N-glycosylamide carbohydrate-peptide bond with LiBH4/tert-BuOH followed by NaBH4-NaOH treatment gave rise to alditols, which were fractionated by means of HPLC. Twelve alditols were isolated in quantities sufficient for the monosaccharide analysis. Possibility of an ovomucoid-type oligosaccharide structure for all the alditols is discussed.     

A rapid preparative method for isolation of neutral and acidic glycosphingolipids by radial thin-layer chromatography

An efficient method to separate neutral and acidic glycosphingolipids (GSLs) from their mixtures within a short period (45-60 min) and with low consumption of solvents (chloroform-methanol-water, 60/35/8 (v/v/v); 250-500 ml) has been developed. This method utilizes a centrifugal thin-layer chromatograph (Chromatotron) and the GSL mixtures (30-400 mg) are applied to glass plates coated with a 1-mm layer of silica gel 60 PF-254. The method (radial thin-layer chromatography) is rapid and simple and the recovery of glycosphingolipids is high (70-80%).     

Purification of beta-glucocerebrosidase by preparative-scale high-performance liquid chromatography: the use of ethylene glycol-containing buffers for chromatography of hydrophobic glycoprotein enzymes

beta-Glucocerebrosidase, partially purified by the method of F. S. Furbish et al. (1977, Proc. Natl. Acad. Sci. USA 74, 3560-3563), was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to contain, in addition to the desired enzyme, variable amounts of a very hydrophobic contaminant (apparent Mr 45,000). Purification of the enzyme was accomplished by gel-permeation HPLC on a TSK 3000 SW column (0.7 X 60 cm). Adsorptive losses of protein on the column were minimized by using buffers containing up to 50% ethylene glycol. We have examined the effects of varying the ethylene glycol concentration on the elution times and recoveries of the two major proteins in this preparation. The high reproducibility of the individual chromatograms permitted the use of an automatic sampler and fraction collector to perform multiple, continuous runs for the purification of milligram quantities of enzyme. Multiple runs of a preparative-scale column, TSK G3000 SWG (2.15 X 60 cm), permitted gram-scale purification of beta-glucocerebrosidase without loss in efficiency of separation. Recovery of enzyme activity is greater than 94% with less than 1% contamination by other proteins. Reaction of enzyme prepared in this fashion with rabbit polyclonal antiserum or mouse monoclonal anti-beta-glucocerebrosidase shows the enzyme to be pure and not immunologically related to the 45,000 Mr contaminant. The specific activity of enzyme prepared by this means is 1.6 X 10(6) nmol/h/mg protein. Inclusion of ethylene glycol in buffers was shown to overcome hydrophobic protein interactions with TSK 3000 SW column matrices for both the soluble human lysosomal enzyme alpha-galactosidase A and the plant toxin ricin.     

A high-performance liquid chromatography approach for isolation and sequencing of chondroitin sulfate oligosaccharides

An apparatus is described which is used for the determination of velocities of falling droplets of small volumes (5 μl) through hydrophobic mixtures. From calibration curves with sodium chloride solutions of different molarities and specific gravities, the densities of the fluid in the droplets were obtained by interpolation. The density of the solutions of proteins may be determined and used in the calculation of partial specific volumes. The hydrophobic fluid used was a mixture of Dow Corning 200, kerosine to reduce the viscosity, and 1,2-dichlorobenzine to increase the specific gravity to approximately that of water.     

Single polyprenol and dolichol isolation by semipreparative high-performance liquid chromatography technique

A new method of separation of single polyprenols (or dolichols) from a mixture of isoprenoid alcohols is described. Application of a high-performance liquid chromatography (HPLC) apparatus equipped with a semipreparative ODS column resulted in preparation of long-chain (dihydro)polyprenols of high purity (>95%).This approach substantially decreases the time scale of the conventional chromatographical preparative procedure. The method can be widely used in chemical and biochemical projects, where single polyprenols or dolichols are required.     

A rapid, isocratic method for phospholipid separation by high-performance liquid chromatography

A rapid, isocratic method for separating the most prevalent phospholipids by high-performance liquid chromatography is described. Baseline resolution of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin is achieved in less than 40 min on a silica column. Lipids are injected in 10 microliter of chloroform-diethyl ether 1:2 (v/v) and eluted with a solvent mixture of acetonitrile-methanol-sulfuric acid 100:3:0.05 (v/v/v) at a flow rate of 1 ml/min. Neutral lipids and cardiolipin elute with the solvent front. Chromatography of a radioactive cell lipid extract indicates a recovery of better than 97%. The procedure is sensitive enough to permit the analysis of the main phospholipids present in a monolayer culture containing about 100 micrograms of cell protein.     

Semipreparative isolation of individual cyanobacterial heterocyst-type glycolipids by reverse-phase high-performance liquid chromatography.

Procedures are described for the quantitative, semi-preparative isolation of individual cyanobacterial heterocyst-type glycolipids (HGs) by reverse-phase HPLC (RP-HPLC) and the modifications to conventional techniques necessary to prevent significant HG losses during sample preparation. Total lipids are obtained from cultures of nitrogen-fixing cyanobacteria by triplicate extraction with 200 packed-cell volumes of chloroform/methanol (1/1, v/v), filtered, and redissolved in chloroform for loading onto a short disposable column of acid-washed silica. After removal of neutral lipids, pigments, and the majority of monogalactosyldiacylglycerol with chloroform and chloroform/acetone, HGs are eluted along with other complex lipids in methanol. The complex lipids are then fractionated by TLC and the HGs isolated as two classes of differing mobilities. The individual components of each class are then resolved by isocratic elution with methanol/water (91/9, v/v) from a C18 RP-HPLC column with refractive index detection. Samples of up to approximately 1.0 mg lipid can be completely separated and the major components isolated in high purity from a single run. Structural studies on the major HG of Nostoc azollae show it to be the glycosylated hexacosane-1,3,25-triol found by others in Anabaena cylindrica.     

A preparative high performance liquid chromatography method for separation of lecithin: comparison to thin-layer chromatography

We have developed a high performance liquid chromatography (HPLC) method to separate lecithin from other phospholipid classes and to obtain lecithin from biologic materials. The separation was performed on a preparative 10-micron Spherisorb column with an optimized solvent system consisting of the following components: acetonitrile, isopropanol, methanol, water, and trifluoroacetic acid. The advantages of this method are the use of an isocratic solvent system limited to about 30 min and the very good separation of the phosphatidyl-choline fraction from the sphingomyelin fraction. Furthermore, the HPLC method has a better recovery rate than the thin-layer chromatography method, and it can be run under automatic control.     

Analytical and preparative high-performance liquid chromatography separation of flavin and flavin analog coenzymes

Reversed-phase high-performance liquid chromatography has been used to separate a number of flavin and flavin analogs at the riboflavin, FMN, and FAD coenzyme level. Analytical methods were developed which enable the facile determination of a particular flavin or mixture of flavins present. These methods also allowed the separation of oxidized from reduced forms of oxygen-stable flavin analogs. Past investigations have utilized enzymatically synthesized FAD analogs with the problem of potential contamination by other levels of the coenzyme or ATP a cosubstrate in the flavokinase/FAD synthetase reaction. Preparative methods show that all the potential reaction products may be separated from one another thereby allowing the rapid purification of these redox coenzyme analogs. To demonstrate the utility of this method, radiolabeled FAD and 1-deazaFAD were prepared and purified.     

Isolation of alpha 1-acid glycoprotein from human plasma using high-performance liquid chromatography

A method for the rapid isolation of purified alpha 1-acid glycoprotein (AGP) from small volumes of human plasma using HPLC has been developed. The method involves preparation of the seromucoid fraction of plasma by sequential perchloric acid and phosphotungstic acid precipitations, followed by chromatography on an HPLC TSKG-3000 column. The yield was high (0.75 mg AGP/ml plasma) and the procedure takes less than 1 day. The method lends itself to easy automation and is particularly suitable for isotopic turnover studies requiring multiple plasma samples.     

A simple preparative method for the isolation of amylose and amylopectin from potato starch

The defatted starch was dispersed in NaOH (1 M) and neutralized with HCl (1 M). The amylose 1-butanol complex is adsorbed on defatted cellulose powder in the solvent system containing acetate buffer (pH 4.8,0.1 M) + urea (2 M) + 1-butanol (8.5%, v/v). The complex adsorbed on cellulose powder is separated by centrifugation (2418 g). The sediment is washed with the solvent system-I to obtain the intermediate fraction. The adsorbed amylose is eluted with urea (2 M) in acetate buffer (pH 4.8, 0.1 M). The amylose, intermediate fraction and amylopectin were precipitated with ethanol, washed free of urea and air dried. They were characterized by determining their blue value and beta -amylolysis limit.     

The isolation of peptides by high-performance liquid chromatography using predicted elution positions

This paper describes the derivation and use of predictive retention coefficients for the reversed-phase high-performance liquid chromatography of peptides. The use of predicted elution positions in the isolation of peptides is illustrated by two examples where peptides, whose existence was postulated from cDNA sequence data, have been successfully isolated. The combination of the powerful chromatographic technology and the ability to predict the elution positions of peptides based on their composition provides a very potent method for the isolation of peptides from biological tissues.     

Determination of acetylmethadol and metabolites by use of high-performance liquid chromatography

A method is described for the simultaneous determination of l,α-acetylmethadol (LAAM) and five active metabolites — noracetylmethadol, dinoracetylmethadol, methadol, normethadol, and dinormethadol — in biofluids by high-performance liquid chromatography using a normal-phase column and a UV detector at 218 nm. The compounds are recovered from biofluids by a multistep liquid—liquid extraction. The mobile phase is methanol—acetonitrile (70:30, v/v) containing 0.015% ammonium hydroxide as the modifier. Retention times can be varied by adjusting the composition of the mobile phase to maximize peak height for quantitation using l-propranolol as the internal standard or peak separation for the collection of fractions. Using a UV detector the lower limit of sensitivity is 10 ng/ml of biofluid. Using fraction collection of radiolabeled drug and metabolites followed by liquid scintillation counting the lower limit of sensitivity is 1.0 ng/ml. Commonly used or abused narcotics including morphine, heroin, meperidine, methadone and propoxyphene do not interfere with the analysis. The method has been applied to plasma and urine samples from humans, sheep and rats. Extracts of urine from patients receiving maintenance treatment with LAAM contain LAAM and each of the five active metabolites.