高效液相色谱法测定雷帕霉素

建立了雷帕霉素高效液相色谱测定方法。其色谱条件：色谱柱Zorbax Eclipse XDB-C8（4.6 mm×150 mm, 3.5 μm）反相柱,以乙腈和水溶液为流动相,紫外检测波长278 nm,流速1.0 mL/min,柱温55 ℃。结果表明：此方法标准曲线范围是0.2～20 μg,线性良好,加样回收率为99.4%～100.79%,日间相对标准偏差（RSD）（n=4）为1.25%～2.25%。本方法操作简便、准确,可有效应用于雷帕霉素提取纯化过程中的检测。     

Purification of human placental acid alpha-mannosidase by an immunological method

An immunoaffinity column was used for the purification of alpha-mannosidase from human placenta. The enzyme was purified to homogeneity by extraction in the presence of various protease inhibitors, immunoaffinity chromatography, Ultrogel AcA-34 gel filtration and hydroxyapatite chromatography. Two subunits were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their molecular weights were 65 kDa and 27 kDa. Heterogeneity of the molecular weight of the large subunit was not observed in our preparation. This method is relatively simple and rapid for obtaining the purified enzyme which is structurally not modified during purification procedures.     

Use of an ALFexpress DNA sequencer to analyze protein-nucleic acid interactions by band shift assay

Gel retardation analysis, or band shift assay, is technically the simplest method to investigate protein-nucleic acid interactions. In this report, we describe a nonradioactive band shift assay using a fluorescent DNA target and an ALFexpress automatic DNA sequencer in place of the current method that utilizes radioactively end-labeled DNA target and a standard electrophoresis unit. In our study, the dsDNA targets were obtained by annealing two synthetic oligonucleotides or by PCR. In both cases, a molecule of indodicarbocyanine (CY5) was attached at the 5' OH end of one of the two synthetic oligonucleotides, with a ratio of one molecule of fluorescent dye per molecule of dsDNA. To demonstrate the feasibility of this new band shift assay method, the DNA-binding proteins selected as models were the BlaI and AmpR repressors, which are involved in the induction of the Bacillus licheniformis 749/I and Citrobacter freundii beta-lactamases, respectively. The results show that the use of an automatic DNA sequencer allows easy gel retardation analysis and provides a fast, sensitive, and quantitative method. The ALFexpress DNA sequencer has the same limit of detection as a laser fluorescence scanner and can be used instead of a FluorImager or a Molecular Imager.     

A new HPLC analytical method for o-hydroxyhippuric acid in uremic serum

o-Hydroxyhippuric acid (HHA) is formed in liver during the process of detoxication of salicylic acid which arises from either salicylic-containing dietary vegetables or hydrolysis of aspirin. Recently, HHA has also been shown as one of the so-called 'uremic toxins'. By derivatization of HHA with o-phthaldialdehyde (OPA), the resulting fluorescent product can easily be measured with the limit of measurement somewhere below 3 pmol on high performance liquid chromatography. Using the method described, an approximately 85% recovery of added HHA in human sera was obtained. This analytical system is now being employed to determine the concentration of HHA in human sera of uremic patients. Determination of HHA content in human uremic patients is very important, since uremia is associated with defective binding of many acidic drugs to serum protein(s) and HHA replaces these drugs by tightly binding to these proteins. This could potentially affect the therapeutic effectiveness of various pharmacologic agents.     

Validation of a reversed-phase HPLC method for quantitative amino acid analysis.

A semi-automated method for amino acid derivatization and analysis has been validated for use in analysis of protein biopharmaceuticals. The method includes protein hydrolysis, o-phthalaldehyde derivatization, and reversed-phase high-performance liquid chromatography analysis in a general-purpose UV-visible high-performance liquid chromatography system. Amino-acid derivatization is performed automatically by the high-performance liquid chromatography autosampler right before injection. The required validation parameters, i.e., specificity, linearity, accuracy, precision, limit of detection, and limit of quantification, were studied for bovine serum albumin and for a recombinant human Fab fragment. The method can be employed as an absolute quantification method for determination of extinction coefficients of recombinant proteins.     

Study of intermolecular interactions and self-organization of adenylic nucleotides by the spin label method

A spin labeling method for obtaining thermodynamic parameters of nucleotide association is proposed. The method is based on the dependence of ESR parameters of the spin-labelled derivative on concentration of the nonlabelled compound due to formation of associates involving both spin-labelled and unmodified molecules. It is found that at pH 7.5 the constant of adenylic nucleotide association practically does not depend on the number of phosphate groups and is equal to 9.7 +/- 0.3 M-1 for AMP, ADP and ATP in 0.1 M NaCl at 28 degrees C. In acidic medium the value of the association constant increases by a factor two. Base stacking is shown to make the main contribution to stability of the associates of adenylic nucleotides at neutral pH, whereas upon base protonation the key role is played, apparently, by base-phosphate interaction. It is thought, that an increase in the solvent entropy is essential for stabilisation of the associates, this factor being more important in the case of nucleotide association as compared to the association of nucleosides. A possible role of nucleotide association in the processes of intracellular regulation is discussed.     

A new electron microscopic method for studying protein-nucleic acid interactions.

A new method for preparation of nucleic acid specimens for electron microscopy has been adapted to study the interaction of proteins with DNA. Both a detergent and a basic protein are added to the DNA-protein solution before spreading on a hypophase containing 0.2 m ammonium acetate. This method has been tested using T7 DNA and Escherichia coli RNA polymerase. Specifically bound enzyme molecules were clearly visible on the well extended DNA molecules; the binding sites were located at 0.59, 1.24, 1.57, and 1.86% of the total length of T7 DNA. Under carefully controlled conditions, 40–85% of the DNA molecules specifically bound at least one enzyme molecule.     

Development of an HPLC method for the determination of nifedipine in human plasma by solid-phase extraction

Nifedipine, a dihydropyridine calcium channel antagonist, is widely used in the treatment of hypertension and other cardiovascular disorders. A selective, sensitive and accurate high-performance liquid chromatographic method has been developed, validated and applied for determination of nifedipine in human plasma samples. A series of studies were conducted in order to investigate the effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers, and to develop a convenient and easy-to-use method for quantitative analysis of nifedipine. The method involves solid-phase extraction on C18 cartridges. The chromatographic separation was accomplished on a Lichrocart Lichrospher 60 RP selectB column with a mobile phase composed of 0.020 mol/L KH2PO4 (pH 4.8) and acetonitrile (42:58, v/v). UV detection was set at 240 nm. The calibration curve was linear in the concentration range of 5.0-200.0 ng/mL for nifedipine in plasma and the limit of quantification was 5.0 ng/mL.     

Amino acid-amino acid interactions studied by charge-transfer chromatography

The interaction between amino acids was studied by charge-transfer reversed-phase thin-layer chromatography. The dependence of the lipophilicity of Trp on the concentration of other amino acids in the eluent was considered to be linearly related to the strength of interaction. Arg, Asn, Glu, Met, Phe and Thr interacted with Trp; Ala, Gly and Ser showed no interaction. Stepwise regression analysis indicated that the pK value of the amino acid side-chain and the lipophilicity of the amino acid had the greatest impact on the interaction, suggesting the simultaneous presence of weak hydrophilic and hydrophobic bonding forces between amino acids. Sodium acetate in the eluent increased the interactive strength between Phe and Trp; acetic acid and sodium chloride did not influence the interaction significantly. No significant difference was found between the effects of l- and d-Asn.     

Quantification of phosphatidic acid and lysophosphatidic acid by HPLC with evaporative light-scattering detection

Phosphatidic acid (PA) and lysophosphatidic acid (LPA) are lipids that regulate cellular processes. PA stimulates kinases and may play a role in exocytosis and membrane fusion. LPA can induce cell proliferation, platelet aggregation, and microfilament formation. Due to the growing interest in these lipids, rapid purification and quantification of these lipids is desirable. We now describe a method that utilizes one HPLC run to separate trace amounts of PA and LPA from large amounts of lipids found in cellular extracts. A two-pump HPLC with a solvent system consisting of chloroform, methanol, water, and ammonium hydroxide was employed to produce a reliable, efficient purification of the two lipids. Lipid mass was quantified by a sensitive evaporative light-scattering detector. Using this new method, insulin addition increased both PA (87%) and LPA (217%) mass in Xenopus laevis oocytes.     

Determination of rat serum esterase activities by an HPLC method using S-acetylthiocholine iodide and p-nitrophenyl acetate

Establishing esterase assays allows the determination and comparison of esteratic activities of tissues of one organism and between organisms. We have developed a high-performance liquid chromatography (HPLC) assay for the determination of S-acetylthiocholine (ATC) and p-nitrophenyl acetate (NPA) hydrolyzing activities of rat serum esterases based on ion pair chromatography with on-line radiochemical and ultraviolet (UV) detection. ATC is a substrate for cholinesterases, whereas NPA is cleaved by a variety of esterases and other proteins (e.g., cholinesterases, paraoxonase, carboxylesterase, albumin). Both substrates were incubated, simultaneously or separately, with rat serum to explore potential interferences between the enzymatic hydrolyses of the compounds. The ratio of the peak area of the ¹⁴C-labeled substrates to the total peak area of the substrates and their corresponding cleavage products was compared with the UV quantitation of ATC and p-nitrophenolate (NP), the cleavage product of NPA, measured at 230 and 350 nm, respectively. The peak identity of ATC and NP was confirmed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The reaction rates of the assays using one substrate or both, as well as using radiochemical or UV detection, were equal. Moreover, the correlation between rat serum volumes and reaction rates was shown for both substrates. In conclusion, one can (i) choose between the two detection methods reliably, (ii) take advantage of monitoring both substrate and product by using radiochemical detection, and (iii) combine both substrates to determine esterase activities in rat serum and probably other biological matrices.     

Analysis of salicylic acid in willow barks and branches by an electrochemical method

An electrochemical method for measuring free salicylic acid (SA) was optimized and used to detect its content in barks and branches of thirteen Salix species. We utilized square wave voltammetry method in combination with pencil lead, the detection limit of which was 1.7 ng/ml of salicylic acid. The highest contents of free SA were observed in the bark of S. laponum (3.0 mg/g fr wt) and in the branches of S. purpurea, cv. Nana (2.1 mg/g fr wt) and S. planifolia (2.2 mg/g fr wt). The technique utilized for determination of SA in willow tissues has a much broader dynamic range and lower limit of detection in comparison to both linear sweep and cyclic voltammetry because of its efficient discrimination of capacitance current. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 623–628. The text was submitted by the authors in English.     

FRET-based analysis of protein-nucleic acid interactions by genetically incorporating a fluorescent amino acid

Protein–nucleic acid interaction is an important process in many biological phenomena. In this study, a fluorescence resonance energy transfer (FRET)-based protein–DNA binding assay has been developed, in which a fluorescent amino acid is genetically incorporated into a DNA-binding protein. A coumarin-containing amino acid was incorporated into a DNA-binding protein, and the mutant protein specifically produced a FRET signal upon binding to its cognate DNA labeled with a fluorophore. The protein–DNA binding affinity was then measured under equilibrium conditions. This method is advantageous for studying protein-nucleic acid interactions, because it is performed under equilibrium conditions, technically easy, and applicable to any nucleic acid-binding protein.