Purification of human placental glucocerebrosidase using a two-step high-performance hydrophobic and gel permeation column chromatography method

Glucocerebrosidase was purified from human placenta approximately 10,600-fold to apparent homogeneity with an overall yield of 37% using cholate extraction, ammonium sulfate fractionation, butanol delipidation, and a two-step high-performance hydrophobic and gel permeation column chromatography method. A Phenyl-5PW (21.5 X 150 mm) column was used in the first step. Approximately one litre of delipidated and dialysed extract containing 3.7 X 10(6) units of enzyme activity from 1 kg of placental tissue was processed by the column at a flow rate of 5 ml/min. Glucocerebrosidase was eluted using a linear cholate gradient (2-3%). There was a 50-fold purification and 89% recovery. The run was completed in about 7 h. In the second step, the concentrated enzyme preparation from the phenyl column was run through two Bio-Sil TSK 250 gel permeation columns (21.5 X 600 mm) connected in series at a flow rate of 1.5 ml/min. A symmetrical peak of glucocerebrosidase activity (Ve = 253 ml) which had constant specific activity (47,000 units/h/mg protein) was noted. There was a 17-fold purification and 80% recovery in this run which was completed in 4 h. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and protein staining with silver compounds of the purified preparation revealed the presence of one band of Mr 68,000.     

Analysis of serum iron by gel permeation high-performance liquid chromatography

A high-performance liquid chromatographic procedure for the determination of serum iron is reported. Serum iron extracted with methyl isobutyl ketone was converted to dibenzoylmethane chelate (molecular weight 725), and it was separated from excess dibenzoylmethane (molecular weight 224) by gel permeation chromatography. The chelate was determined by measuring ultraviolet absorption at 280 nm. Good reproducibility, recovery, and correlation with the conventional colorimetric method were observed.     

Purification of radiolabeled and native polypeptides by gel permeation high-performance liquid chromatography

Some results and observations concerning the use of protein columns are presented. The combined use of four protein columns having different fractionation ranges together with a volatile triethylamine formate buffer allowed the sieving of various polypeptides according to their molecular weights over a range of 500 to 150,000. The addition of 4 or 6 m guanidine-HCl permitted the reduction of aggregation with no sacrifice in resolution or linearity. With that denaturant, rapid separation, and molecular weight determination in the range 500–90,000 is easily accomplished. Moreover, sample recoveries as determined with radiolabeled proteins always exceeded 70% while radioimmunoassay techniques can be directly applied to the column eluate. Applications to quick identification of natural fragments of a serine protease, tonin, analysis of maturation products of pro-opiomelanocortin in an in vitro pulse experiment and finally quantitation by radioimmunoassays of pituitary peptides and elution of their ¹²⁵I-labeled derivatives are described.     

Determination of concentrations of flecainide in human serum by high-performance liquid chromatography on a fluorocarbon-bonded silica gel column

An optimized method for the determination of flecainide in serum is presented. Extraction using a solid-phase C₁₈ column and chromatography on a stabilized fluorocarbon-bonded silica gel column effectively separate flecainide from an internal standard (a positional isomer of flecainide). The HPLC apparatus and conditions were as follows: analytical column, Fluofix 120N; sample solvent, 20 μl; column temperature, 40°C; detector, Shimadzu RF-5000 fluorescence spectrophotometer (excitation wavelength=300 nm, emission wavelength=370 nm); mobile phase, 0.06% phosphoric acid containing 0.1% tetra-n-butyl ammonium bromide–acetonitrile (75:25, v/v); flow-rate, 1.0 ml/min. The standard curves for flecainide were linear in the concentration range examined (10–2000 ng/ml). The regression equation was y=0.08+0.0078x (r=0.9998). The minimum detectable amount of flecainide was approximately 5 ng/ml. In the within-day study, the precision coefficients of variation were 2.66, 2.18, 2.54, 2.72, 2.88, 2.24, and 3.29% for the 10, 50, 100, 200, 500, 1000, and 1500 ng/ml standards, respectively. The absolute recovery rates of flecainide at each concentrations were 94–100%. The method described provides analytical sensitivity, specificity and reproducibility suitable for both biomedical research and therapeutic drug monitoring.     

High-performance liquid chromatography of proteins by gel permeation chromatography

The ability of two high-performance liquid chromatography gel permeation columns to separate proteins was evaluated. These columns gave satisfactory molecular weight separations for some, but not all, proteins tested. These results indicate that there are limitations in confidence of molecular weight determinations made by this technique.     

Simple purification of aromatic L-amino acid decarboxylase from human pheochromocytoma using high-performance liquid chromatography

We purified aromatic L-amino acid decarboxylase (AADC) homogeneously and rapidly from human pheochromocytoma using high-performance liquid chromatography. HPLC with gel permeation and hydrophobic columns was highly effective, and the entire purification could be finished within 3 days. Purified AADC showed a single band with an Mr of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and decarboxylated L-3,4-dihydroxyphenylalanine, L-5-hydroxytryptophan, and L-threo-3,4-dihydroxyphenylserine (a synthetic precursor of natural norepinephrine). Amino acid analysis of purified AADC was performed.     

Simple purification of aromatic -amino acid decarboxylase from human pheochromocytoma using high-performance liquid chromatography

We purified aromatic -amino acid decarboxylase (AADC) homogeneously and rapidly from human pheochromocytoma using high-performance liquid chromatography. HPLC with gel permeation and hydrophobic columns was highly effective, and the entire purification could be finished within 3 days. Purified AADC showed a single band with an M_r of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and decarboxylated -3,4-dihydroxyphenyl-alanine, -5-hydroxytryptophan, and -threo-3,4-dihydroxyphenylserine (a synthetic precursor of natural norepinephrine). Amino acid analysis of purified AADC was performed.     

Determination of opipramol in human plasma by high-performance liquid chromatography with photometric detection using a cyanopropyl column

A high-performance liquid chromatographic method is described for the determination of opipramol in human plasma. Opipramol was extracted into tert.-butylmethyl ether, separated on a cyanopropyl silica column and detected at 254 nm. Imipramine was used as internal standard. The limit of quantitation was 250 pg/ml using 1.5 ml plasma. Precision was better than 9%, inaccuracy less than 8%. The assay is more sensitive than previously published methods, and it has been applied to the analysis of plasma samples from a pharmacokinetic study.     

Preparative-scale purification of RNA using an efficient method which combines gel electrophoresis and column chromatography.

Here we describe a reliable method for purifying large amounts of RNA of any sequence and length with comparable efficiency and resolution of gel electrophoresis and with capacity approaching that of column chromatography. The RNA mixture of interest is separated on a cylindrical denaturing polyacrylamide gel, eluted by a peristaltic pump, detected by a UV-vis detector, and collected by a fraction collector. Using this method, we were able to separate one third of a 100 ml in vitro transcribed 34mer hammerhead ribozyme (approximately 6.2 mg) in a single run. The entire 100 ml transcribed RNA (approximately 18.5 mg) was separated after consecutive runs using one single gel preparation.     

A rapid high-performance liquid chromatography purification method of lodinated polypeptide hormones

A reverse-phase high-performance liquid chromatography procedure is presented for the separation of a variety of monoiodinated peptides from the corresponding unlabeled and diiodinated hormones. In the case of peptides lacking Met residues, susceptible to oxidation by the chloramine-T-labeling method, chloramine T treatment alone had no effect on elution position of unlabeled hormone. For those hormones containing Met, however, more than 90% of the peptide was transformed into the earlier-eluting oxidized form after 20 to 40 s of chloramine-T treatment. Upon iodination, monoiodinated derivatives were well separated from the corresponding chloramine-T-treated standards, the extent of separation being decreased with increased molecular weight. Application of this technique to the purification of monoiodinated ¹²⁵I-γ-melanocyte-stimulating hormone permitted a threefold increase in titer and sensitivity of a γ-melanocyte-stimulating hormone radioimmunoassay system.     

Purification of human platelet membrane glycoproteins IIb and IIIa using high-performance liquid chromatography gel filtration

A method for purifying platelet membrane glycoproteins IIb and IIIa to homogeneity has been developed. The procedure involves high-pressure gel filtration chromatography using a TSK-4000SW column in the presence of sodium dodecyl sulfate. This technique is capable of rapidly preparing milligram quantities of each glycoprotein with greater than 90% recovery. The use of this technique should aid in defining the structural and functional properties of GPIIb and GPIIIa.     

Molecular weight determination of peptides by high-performance gel permeation chromatography

A method for molecular weight determination of small peptides using Bio-Sil TSK 20 and Bio-Gel TSK 125 columns is described. The TSK 20 column provided a good separation of the standard peptides in the range from 1000-10,000 with an accuracy of less than 5% from the calculated regression line. Two combined TSK 125 columns allowed a reliable molecular weight determination in the range from 800 to 3500.     

Determination of oxytetracycline levels in rainbow trout serum on a biphenyl column using high-performance liquid chromatography

We developed a simple and sensitive high-performance liquid chromatography method on a biphenyl column to determine oxytetracycline (OTC) levels in rainbow trout serum. The assay used deproteination, filtration, and subsequent separation on a reverse-phase biphenyl column, with UV detection at 355 nm. OTC (7.8-7.9 min) was completely resolved from structurally similar riboflavin (10.4-10.5 min), a common feed supplement. Estimated limits of detection and quantitation of OTC were 0.01 and 0.04 microg/mL, respectively. The average recovery for OTC was 102% with a R.S.D. of 8.34%. Calibration standards were linear from 0.01 to 10 microg/mL.     

On-line sample preparation of paraquat in human serum samples using high-performance liquid chromatography with column switching

A new ion-pair high-performance liquid chromatographic method with column-switching has been developed for the determination of paraquat in human serum samples. The diluted serum sample was injected onto a precolumn packed with LiChroprep RP-8 (25-40 μm) and polar serum components were washed out by 3% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 5 mM sodium octanesulfonate. After valve switching to inject position, concentrated compounds were eluted in the back-flush mode and separated on an Inertsil ODS-2 column with 17% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 10 mM sodium octanesulfonate. The total analysis time per sample was about 30 min and mean recovery was 98.5±2.8% with a linear range of 0.1–100 μg/ml. This method has been successfully applied to serum samples from incidents by paraquat poisoning.     

Rapid purification of synthetic oligonucleotides: a convenient alternative to high-performance liquid chromatography and polyacrylamide gel electrophoresis

A new method has been developed to purify and detritylate milligram amounts of synthetic oligonucleotides. Dimethoxytrityl oligonucleotides from 15 to 100 nucleotides in length are applied in triethylammonium acetate or concentrated ammonium hydroxide to a disposable chromatographic cartridge, the NENSORB PREP Nucleic Acid Purification Cartridge. Salts, failure sequences and synthetic by-products are washed away while the desired, full-length, dimethoxytrityl oligonucleotide remains bound to the cartridge. The trityl group is hydrolyzed from the 5'-end of the oligonucleotide with an acid wash and then the purified oligonucleotide is eluted with 35% methanol. Oligonucleotides are recovered salt-free with purities greater than 95%. NENSORB PREP-purified primers provide superior sequence data compared to similar primers used without purification and equivalent data to primers purified by polyacrylamide gel electrophoresis when used in manual radiometric Sanger sequencing.     

Histamine-releasing activity. V. Characterization and purification using high-performance liquid chromatography

The production of large quantities of the lymphokine(s) histamine-releasing activity (HRA) and its partial purification by Sephadex G-75 and ion-exchange chromatography on carboxymethyl (CM) Sepharose 6B have been detailed (M. A. Lett-Brown, D. O. Thueson, D. E. Plank, M. P. Langford, and J. A. Grant, Cell. Immunol. 87, 434-444, 1984). Two peaks of activity (HRA I and II) were recovered. Preparations of HRA have now been analyzed by high-performance liquid chromatography (HPLC). Thoracic duct lymphocytes stimulated with 200 U/ml streptokinase were used as a source of HRA. Gel-filtration HPLC on a TSK 3000 column separated HRA into two peaks of activity (10,000-20,000 and 1300 Da). Reverse-phase high-performance liquid chromatography using a Nucleosil C-8 column showed that HRA II (the activity eluted at a conductivity of 18-20 mmho on the CM-Sepharose column) eluted as a single sharp peak, the main protein contaminant being cytochrome c, the carrier protein added to enhance the yield of HRA. High-performance liquid chromatography was found to be a useful analytical tool and may be suitable for the large-scale purification of HRA.     

Simple method for the analysis of tenoxicam in human plasma using high-performance liquid chromatography

A simple, rapid and cost-effective method for the determination of tenoxicam in human plasma is described, using ketorolac as the internal standard. The extraction procedure utilised 5% zinc sulphate and methanol. A nucleosil C₁₈ column and 35:65 acetonitrile-water phosphate buffered mobile phase (pH 2.8) were used, with ultraviolet detection at 355 nm. The assay was linear in the range 40 ng/ml-10 μg/ml, with recovery of extraction ranging from 87 to 102%. The intra- and inter-assay reproducibility had coefficients of variation of 3.9–7.7 and 1.6% respectively. The limit of detection for this method was 40 ng/ml.     

Rapid, quantitative high-performance liquid column chromatography of pseudouridine

A rapid, precise, and accurate chromatographic method for the determination of pseudo-uridine (ψ) in urine by high-performance liquid chromatography (HPLC) has been developed. The ribonucleosides were first isolated with an affinity gel containing immobilized phenylboronic acid. The response for ψ was linear well above and below the range necessary to determine urinary ψ. Good precision was obtained for both matrix-dependent and matrix-independent samples. Supporting experimental data are presented on precision, recovery, chromatographic methods, sample cleanup and application to the analysis of urine samples from normal males and females, and patients with advanced colon cancer. In a comparison of 40 normals with 10 colon cancer patients, 9 of the 10 patients had a ψ: creatinine (Cr) ratio greater than + 2σ for the normal population. This HPLC method is now being used extensively in our laboratory as a routine method for determination of ψ in urine from patients with various types of cancer and in chemotherapy response studies. Data are presented on the dynamics of ψ excretion by normal males and females. When the excretion of ψ was normalized with the excretion of creatinine, it was noted that samples collected at random have the same ψ: Cr ratio value as for the 24-h total collection, thus, allowing the use of random samples. The constancy of the ψ: Cr ratio implies that RNA turnover is constant and ψ excretion is independent of diet. Base values are presented for the ψ: Cr