Separation of complexes containing protein A and IgG or Fcγ fragments by high-performance liquid chromatography

Protein A of Staphylococcus aureus is a bivalent Fc receptor that can form complexes with immunoglobulin G (IgG) or Fcγ fragments that activate humoral (e.g., complement) and cellular (e.g., lymphocyte) components of the immune system both in vitro and in vivo. To obtain complexes formed between protein A of Staphylococcus aureus (SpA) and rabbit IgG or Fcγ fragments for purposes of characterizing their compositions and studying their biological activities, we have used high-performance liquid chromatography to separate complexes in 20 min. Complexes were prepared with trace amounts of ¹²⁵I-SpA and ¹³¹I-IgG or ¹³¹I-Fcγ to simplify the analyses. With excess molar amounts of IgG or Fcγ the complexes have the molecular formulas [(IgG)₂SpA]₂ or [(Fcγ)₂SpA]₂. With excess SpA, complexes corresponding to (IgG)(SpA) or (Fcγ)(SpA) are formed, perhaps with other complexes that have different ratios of components. Since SpA is a rod-shaped molecule it elutes at a molecular weight corresponding to 240,000 rather than the true value of 42,000. This behavior is reflected in the elution of certain complexes at shorter retention times than expected on the basis of actual molecular weights, and facilitates separation of complexes from free IgG or Fcγ. The true molecular weights and molecular formulas of complexes isolated by HPLC were verified by ultracentrifugation. This HPLC method was used to study the interconversion and stability of complexes.     

Determination of domperidone in human serum and human breast milk by high-performance liquid chromatography–electrospray mass spectrometry

A sensitive and selective method for the determination of domperidone in human breast milk and serum has been developed. The same method may be successfully applied to both matrices to a lower limit of quantitation of 0.5 ng/ml. Samples are processed by a liquid–liquid extraction, and analyzed by LC–ESI-MS in positive ion mode. There was no interference, on the domperidone quantitation, from over 30 drugs. Samples from patients, at various times post-dose, were analyzed and a large number showed significant levels of domperidone in the breast milk as well as in the serum.     

The purification of TEM-β-lactamase from cell extract using immobilized copper affinity chromatography

Summary TEM--lactamase was purified by immobilized metal affinity chromatography from E. coli cell extracts. It was hypothesized that this protein could be purified in one step due to the abundence of metal binding residues. A pH gradient eluted the cell extract proteins. EDTA treatment released TEM--lactamase. This procedure is much simpler than the current methods employed for TEM--lactamase purification.     

Method for measurement of the quaternary amine compounds paraquat and diquat in human urine using high-performance liquid chromatography–tandem mass spectrometry

We have developed a highly selective and sensitive analytical method to quantify paraquat and diquat by use of high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS). The sample preparation includes solid phase extraction that uses weak cation exchange cartridges. These highly charged dual quaternary amines were not retained by standard reversed phase columns, but they could be adequately separated through HPLC with a HILIC column. The detection was carried out with a triple quadrupole mass spectrometer with an electrospray ionization probe in positive ion mode in multiple reaction monitoring. Repeated analysis in human urine samples spiked with low (5 ng/ml), medium (15 ng/ml), and high (30 ng/ml) concentrations of the analytes yielded relative standard deviations of less than 9%. The extraction efficiencies ranged from 77.7% to 94.2%. The limits of detection were in the range of 1 ng/ml.     

Determination of pyridoxal 5′-phosphate as the semicarbazone derivative using high-performance liquid chromatography

A high-performance liquid chromatographic (hplc) procedure is described for the determination of pyridoxal 5′-phosphate (PLP) in animal tissues. The procedure is based on extraction with perchloric acid and treatment with semicarbazide to form PLP-semicarbazone. This derivative is quantitatively determined using hplc with an octylsilica column, an acidic phosphate mobile phase buffer, and fluorometric detection. The validity of the method was confirmed by inspection of the fluorescence spectra of the PLP-semicarbazone hplc peaks of semicarbazide-treated standards and tissue extracts. Preparative chromatography for extract purification is not required because of the hplc efficiency and detection specificity. This method provides a simple technique for the rapid, direct assay of PLP in animal tissues.     

Quantification of dialkylphosphate metabolites of organophosphorus insecticides in human urine using 96-well plate sample preparation and high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry

Organophosphorus (OP) pesticides kill by disrupting a targeted pest's brain and nervous systems. But if humans and other animals are sufficiently exposed, OP pesticides can have the same effect on them. We developed a fast and accurate high-performance liquid chromatography–tandem mass spectrometry method for the quantitative measurement of the following six common dialkylphosphate (DAP) metabolites of organophosphorus insecticides: dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate, (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP). The general sample preparation included 96-well plate solid phase extraction using weak anion exchange cartridges. The analytical separation was performed by high-performance liquid chromatography with a HILIC column. Detection involved a triple quadrupole mass spectrometer with an ESI probe in negative ion mode using multiple reaction monitoring. Repeated analyses of urine samples spiked at 150, 90 and 32 ng/mL with the analytes gave relative standard deviations of less than 22%. The extraction efficiency ranged from 40% to 98%. The limits of detection were in the range of 0.04–1.5 ng/mL. The throughput is 1152 samples per week, effectively quadrupling our previous throughput. The method is safe, quick, and sensitive enough to be used in environmental and emergency biological monitoring of occupational and nonoccupational exposure to organophosphates.     

Removal of the ϵ subunit from Escherichia coli F1-ATPase using monoclonal anti-ϵ antibody affinity chromatography

The usefulness of two monoclonal antibodies, ϵ-1 and ϵ-4, which recognize the ϵ subunit of Escherichia coli F₁-ATPase, for removing that subunit from ATPase was assessed. The ϵ subunit is a tightly bound, but dissociable, inhibitor of the ATPase. ϵ-1 binds ϵ with 10-fold higher affinity than ϵ-4. ϵ-1 recognizes a site on ϵ which is hidden by the quaternary structure of ATPase, while ϵ-4 can recognize ϵ when it is part of ATPase. Each antibody was purified and coupled to Sepharose to generate affinity columns. Solutions of ATPase in a buffer which was designed to reduce the affinity of ϵ for the enzyme were pumped through the columns and the degree of ϵ depletion was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blotting. Neither column retained ATPase significantly. At low ATPase concentrations and low flow rates, the ϵ-1 column was more efficient than the ϵ-4 column, removing in excess of 95% of the ϵ in a single passage compared with 93% removal by the ϵ-4 column. At higher protein concentrations or flow rates, however, the performance of the ϵ-1 column was substantially poorer, while that of the ϵ-4 column was much less affected. Very little ϵ emerged from the ϵ-4 column before most of the measured ϵ-binding capacity was filled. A second passage through the ϵ-4 column reduced residual ϵ to less than 2% of that which was originally present. Pure, active ϵ was eluted from either column by 1 m NH₄OH, pH 11. The relatively poor performance of ϵ-1 is discussed in terms of the low availability of the epitope and the tendency of the ϵ-depleted complex to compete with ϵ-1 for residual ϵ subunit. From consideration of these factors it appears likely that antibodies which recognize exposed epitopes will generally be more effective than antibodies which recognize cryptic epitopes in removing spontaneously dissociable subunits from protein complexes.     

Determination of imidapril and imidaprilat in human plasma by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive and specific assay of imidapril and its active metabolite, imidaprilat, in human plasma has been developed. This method is based on rapid isolation and high-performance liquid chromatography (HPLC)–electrospray ionization (ESI)-tandem mass spectrometry (MS–MS). Imidapril and imidaprilat were isolated from human plasma using OASIS HLB (solid-phase extraction cartridge), after deproteinization. The eluent from the cartridge was evaporated to dryness, and the residue was reconstituted in mobile phase and injected into the HPLC–ESI-MS–MS system. Each compound was separated on a semi-micro ODS column in acetonitrile–0.05% (v/v) formic acid (1:3, v/v). The selected ion monitoring using precursor→product ion combinations of m/z 406→234 and 378→206, was used for determination of imidapril and imidaprilat, respectively. The linearity was confirmed in the concentration range of 0.2 to 50 ng/ml in human plasma, and the precision of this assay, expressed as a relative standard deviation, was less than 13.2% over the entire concentration range with adequate assay accuracy. The HPLC–ESI-MS–MS method correlates well with the radioimmunoassay method, therefore, it is useful for the determination of imidapril and imidaprilat with sufficient sensitivity and specificity in clinical studies.     

Improved purification of α-amylase isolated fromRhizomucor pusillus by affinity chromatography

A highly purified extracellular -amylase was isolated fromRhizomucor pusillus with minimum loss of enzymatic activity. The enzyme was purified from the mycelium-free liquid filtrate of the thermophilic moldRhizomucor pusillus. Maximum enzyme yields were attained after 5 days of growth on liquid starch-yeast extract at 45°C and pH 7.0. The crude enzyme preparation was first concentrated 80-fold by ultrafiltration. Purification was recently achieved with high-performance liquid chromatography and Waters Protein Pak 300 SW. Improved purification was then achieved with a dextrin-bound affinity column, with a 59-fold increase in specific activity from the crude enzyme preparation. This final enzyme preparation produced a single band on polyacrylamide gel electrophoresis. The molecular weight determined by SDS gel electrophoresis was 52,000 daltons.     

Expression and purification of moricin CM4 and human β-defensins 4 in Escherichia coli using a new technology

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human β-defensins 4 (HβD4) in the Escherichia coli. The CM4 and HβD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HβD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide.     

Detection of landiolol using high-performance liquid chromatography/fluorescence: A blood esterase-sensitive ultra-short-acting β1-receptor antagonist

Landiolol hydrochloride, a new adrenergic β₁-selective antagonist having an ultra-short half-life, is used to prevent tachyarrhythmia during surgery. Since landiolol is thought to be rapidly hydrolyzed to an inactivate metabolite by esterases, quantification of the drug concentration in the blood is impractical. The landiolol concentration in blood was halved within 5 min after blood sampling. This degradation was effectively prevented by pre-treatment with neostigmine (100 μg) in the sampling tube, but not by EDTA pre-treatment, indicating that landiolol could be metabolized by pseudocholinesterase in plasma. After the one-step solid-phase extraction, fluorescence detection of landiolol reduced chromatographic background signals and then improved assay sensitivity to the lower limit of 10 ng/ml in blood; this reproducible approach yielded coefficient variation of less than 6%. The blood concentration-time profile of landiolol hydrochloride in patients of the present investigation afforded more practical assessment than previously reported studies, thus improving accuracy and facilitating detailed pharmacokinetic study in relation to the pharmacological action of drug.     

Separation and quantification of d- and l-phosphoserine in rat brain using Nα-(2,4-dinitro-5-fluorophenyl)-l-alaninamide (Marfey's reagent) by high-performance liquid chromatography with ultraviolet detection

d-Serine has recently been described to be present in the brain at high concentrations. However, while prior research has demonstrated that l-phosphoserine is the major precursor of l-serine in the brain, the possible role of d-phosphoserine as the direct precursor of d-serine is unknown. To address this problem, we developed an assay to separate and quantitate d- and l-phosphoserine. A very simple HPLC-UV procedure for the separation and quantification of d- and l-phosphoserine is presented using precolumn derivatization with a chiral reagent, Nα-(2,4-dinitro-5-fluorophenyl)-l-alaninamide (Marfey's reagent), and a conventional C₁₈ reversed-phase column. The procedure is sensitive to 11 pmol on-column and derivatives are stable for at least two weeks at room temperature. Rat brain regions (cortex, hippocampus, striatum, and cerebellum) were analyzed for the presence of d- and l-phosphoserine. It was determined that the brain regions studied contained exclusively l-phosphoserine.     

Refolding and purification of recombinant human interferon-γ expressed as inclusion bodies inEscherichia coli using size exclusion chromatography

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human interferon-γ (rhIFN-γ) at a high concentration. The rhIFN-γ was overexpressed inE. coli, resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the bufferexchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded rhIFN-γ, with protein recovery of 67.1% and specific activity up to 1.2×10⁷ IU/mg.     

Development of tryptophan-modified human serum albumin columns for site-specific studies of drug–protein interactions by high-performance affinity chromatography

Human serum albumin (HSA) is one of the main proteins involved in the binding of drugs and small solutes in blood or serum. This study examined the changes in chromatographic properties that occur for immobilized HSA following the chemical modification of HSA's lone tryptophan residue (Trp-214). Trp-214 was reacted with o-nitrophenylsulfenyl chloride, followed by immobilization of the modified protein and normal HSA onto separate silica-based HPLC supports. The binding properties of the modified and normal HSA were then analyzed and compared by using frontal analysis and zonal elution experiments employing R/S-warfarin and l-tryptophan as probe compounds for the warfarin and indole binding regions of HSA. The modified HSA was found to have the same number of binding sites as normal HSA for R-warfarin and l-tryptophan but lower association equilibrium constants for these test solutes. Zonal elution studies with R- and S-warfarin on the modified HSA column demonstrated the importance of Trp-214 in determining the stereoselective binding of HSA for these agents. These studies also indicated that tryptophan modification can alter HSA-based separations for chiral solutes.     

Determination of 17α-dihydroequilenin in rat,rabbit and monkey plasma by high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the determination of total (unconjugated and conjugated) 71α-dihydroequilenin in male and female rat rabbit and male rhesus monkey plasma is described here. Plasma sample preparation involved hydrolysis with enzyme (Glusulase), addition of internal standard (14β-equilenin) and solvent extraction. The extracts were chromatographed on a C₆, 5-μm reversed-phase HPLC column and detection was accomplished with a fluorescence detector operated at an excitation wavelength of 210 nm and an emission wavelength of 370 nm. The assay was linear over a range of 2.5 to 100 ng/ml in male and female rat plasma, and 5 to 500 ng/ml in female rabbit and male and female monkey plasma. The method was specific, accurate and reproducible (percent differences <14.5; coefficients of variation <9.5%) in all matrices examined. The applicability of this method was successfully tested by quantifying total plasma concentrations of 17α-dihydroequilenin in ovariectomized female rats, ovariectomized female rabbits and a normal female rhesus monkey receiving 2.0, 8.3 and 0.1 mg/kg, respectively, of 17α-dihydroequilenin sulfate intragastrically.     

Two-step chromatographic purification of human plasma α1-acid glycoprotein Its application to the purification of rare phenotype samples of the protein and their study by chromatography on immobilized metal chelate affinity adsorbent

α₁-Acid glycoprotein (AAG) or orosomucoid was purified to homogeneity from human plasma by a separate two-step method using chromatography on immobilized Cibacron Blue F3G-A to cross-linked agarose and chromatography on hydroxyapatite. The conditions for the pre-purification of AAG by chromatography on immobilized Cibacron Blue F3G-A were first optimized using different buffer systems with different pH values. The overall yield of the combined techniques was 80% and ca. 12 mg of AAG were purified from an initial total amount of ca. 15 mg in a ca. 40 ml sample of human plasma. This method was applied to the purification of AAG samples corresponding to the three main phenotypes of the protein (F1^s*S/A, F1/A and S/A), from individual human plasma previously phenotyped for AAG. A study by isoelectric focusing with carrier ampholytes showed that the microheterogeneity of the purified F1^s*S/A, F1/A and S/A AAG samples was similar to that of AAG in the corresponding plasma, thus suggesting that no apparent desialylation of the glycoprotein occurred during the purification steps. This method was also applied to the purification of AAG samples corresponding to rare phenotypes of the protein (F1/A¹AD, S/A¹X₀ and F1/A¹C1) and the interactions of these variants with immobilized copper(II) ions were then studied at pH 7, by chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the different variants encoded by the first of the two genes coding for AAG in humans (i.e. the F1 and S variants) interacted non-specifically with the immobilized ligand, whereas those encoded by the second gene of AAG (i.e. the A, AD, X₀ and C1 variants) strongly bound to immobilized Cu(II) ions. These results suggested that chromatography on an immobilized affinity Cu(II) adsorbent could be helpful to distinguish between the respective products of the two highly polymorphic genes which code for human AAG.