用质粒作底物测定核糖体失活蛋白酶活性的新方法

植物核糖体失活蛋白(ribosome-inactivating protein,RIP)是一类能作用于核糖体最大RNA的独特蛋白质.它是研究蛋白质生物合成中核糖体RNA结构与功能的有力工具.利用RIP能在DNA中脱去一些腺嘌呤碱基使超螺旋DNA解旋的特点,分别以常用的质粒PUC18、PUC19和PBR322 DNA为底物,建立了测定RIP酶活性的一种新方法,其灵敏度是50ng(天花粉蛋白)和5ng(还原型的辛纳毒蛋白),酶催化反应的时间是60min.这个新方法具有方便、快捷、灵敏的特点,避免了常用方法中制备核糖体、提取RNA的仪器和技术条件的限制,检测的时间由原来的几天缩短到约120min,大大地降低了检测的费用,为广泛和深入地研究RIP提供了有利的条件.     

Liquid chromatographic method for the determination of plasma itraconazole and its hydroxy metabolite in pharmacokinetic/bioavailability studies

A sensitive and selective high-performance liquid chromatographic method was developed for the determination of itraconazole and its active metabolite, hydroxyitraconazole, in human plasma. Prior to analysis, both compounds together with the internal standard were extracted from alkalinized plasma samples using a 3:2 (v/v) mixture of 2,2,4-trimethylpentane and dichloromethane. The mobile phase comprised 0.02 M potassium dihydrogen phosphate-acetonitrile (1:1, v/v) adjusted to pH 3.0. Analysis was run at flow-rate of 0.9 ml/min with excitation and emission wavelengths set at 260 and 365 nm, respectively. Itraconazole was found to adsorb on glass or plastic tubes, but could be circumvented by prior treating the tubes using 10% dichlorodimethylsilane in toluene. Moreover, rinsing the injector port with acetonitrile helped to overcome any carry-over effect. This problem was not encountered with hydroxyitraconazole. The method was sensitive with limit of quantification of 3 ng/ml for itraconazole and 6 ng/ml for hydroxyitraconazole. The calibration curve was linear over a concentration range of 2.8-720 ng/ml for itraconazole and 5.6-720 ng/ml for the hydroxy metabolite. Mean recovery value of the extraction procedure for both compounds was about 85%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 15%. Hence, the method is suitable for use in pharmacokinetic and bioavailability studies of itraconazole.     

Quantitation of surfactant protein B by HPLC in bronchoalveolar lavage fluid

A sensitive reversed phase HPLC method with evaporative light scattering detection (ELSD) was developed for the determination of the hydrophobic surfactant protein B (SP-B) in human bronchoalveolar lavage fluid. Samples were extracted two times with CHCl(3):MeOH:HCl (2:3:0.005N) solution in a ratio of 1:2 by volume. The extract of the lower phase was separated on a C4 butyl silica gel column with an isocratic elution using a mobile phase, consisting of 97% methanol, 2.75% chloroform and 0.25% 0.1 M trifluoroacetic acid (by volume), at a flow rate of 1 ml/min. SP-B was detected by ELSD and quantified by comparison to an external standard. The duration of a run was 7 min, the quantification limit 30 ng and the limit of detection was at about 15 ng of SP-B. This method is suitable for the rapid routine quantification of SP-B in human bronchoalveolar lavage fluid samples.     

Improved high-performance liquid chromatographic analysis of terazosin in human plasma

A simple, sensitive and reproducible high-performance liquid chromatography (HPLC) method was developed for the determination of terazosin in human plasma. The method involves a one-step single solvent extraction procedure using dichloromethane with a 0.25 ml plasma sample. Recovery values were all greater than 90% over the concentration range 0.25–100 ng/ml. Terazosin was found to adsorb to glass or plastic tubes, but this could be circumvented by using disposable plastic tubes. Also, rinsing the injector port with methanol after each injection helped to prevent any carry-over effect. The internal standard, prazosin, did not exhibit this problem. The method has a quantification limit of 0.25 ng/ml. The within- and between-day coefficient of variation and accuracy values were all less than 7% over the concentration range 0.25–100 ng/ml and hence the method is suitable for use in pharmacokinetic studies of terazosin.     

Previsible silver staining of protein in electrophoresis gels with mass spectrometry compatibility

A convenient silver staining method for protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8 ng/band in approximately 1 h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1 h with a detection limit of 0.2 ng/band.     

Detection of proteases by clotting of casein after gel electrophoresis

Clotting of casein provides a sensitive method for detection of proteases after gel electrophoresis. The method is here designated "caseogram." After electrophoresis the gel was equilibrated with 0.15-0.3 M sodium acetate, pH 5.3, and an 1% agarose gel containing 1% skim-milk powder in 0.1 M sodium acetate, pH 5.3, was placed on top of the electrophoresis gel. By incubation at 37 degrees C for 2 h the protease-containing zones produced distinct precipitates in the skim-milk gel. For permanent documentation the skim-milk gel was stained with amido black. The detection limit for pepsin A is 5 ng in the caseogram against 25 ng by hemoglobin digestion at pH 2.5. For calf chymosin it is 1 ng against 100 ng by digestion of hemoglobin at pH 3.5. Caseograms work well after agar gel electrophoresis, after different types of immunoelectrophoresis, and after isoelectric focusing or disc electrophoresis in polyacrylamide gels. Since inert proteins do not interfere with the detection, the method is especially suitable for analysis of crude samples. Samples containing pepsinogen or pepsinogen-like zymogens may be activated at pH 2 before equilibration at pH 5.3.     

Determination of Oltipraz in serum by high-performance liquid chromatography with optical absorbance and mass spectrometric detection

Three methods have been developed for the analysis of Oltipraz in serum. A method suitable for routine use employs spiking with a homologous internal standard, off-line solid-phase extraction, high-performance liquid chromatographic separation, and optical absorbance detection at 450 nm. Method detection limit is about 1 ng/ml. A second method, less susceptible to bias from co-eluting interferences, uses a stable isotope-labeled internal standard, similar extraction and separation, and detection by thermospray mass spectrometry. Method detection limit is about 0.2 ng/ml. A third method was developed which can be used without specially synthesized internal standards. It uses on-line solid-phase extraction, with quantification by comparison with external standards. Method detection limit is about 3 ng/ml. Good agreement was observed between these methods and with similar and different methods run in other laboratories. Calibration curves were linear over the entire range which was investigated, i.e., up to 500 ng/ml. Coefficients of variation were similar for all three methods, being about 5%.     

General proteinase assay by formation of SDS-protein gels of proteolyzed substrate proteins

A new approach to the assay of proteinases is described. The method relies on water-insoluble protein substrates, such as gluten and fibrin, which form expanded gels in the presence of sodium dodecyl sulfate (SDS) reagent. Powdered substrate is dispersed in buffer and aliquots are pipetted into long, narrow, 400-microliters tubes made of clear polypropylene. After the addition of enzyme and a period of incubation, a SDS reagent is added, the tubes are centrifuged, and the height of the SDS-protein gel is measured. Reduction of gel height gives a direct measure of enzyme activity. Salt concentration, pH, and incubation times must be consistent for both test and control reactions in order to obtain reproducible results. Examples of proteinases measured by this method are trypsin, chymotrypsin, elastase, pronase, papain, pepsin, an insect (Nysius huttoni) salivary proteinase, and wheat proteinase. The assay could detect enzyme in crude extracts or in purified form. In 1-h incubations, 10 ng of pepsin and elastase or 20 ng of purified insect proteinase could be detected. The assay was simple, fast, economical, and sensitive.     

Single-step electroelution of proteins from SDS-polyacrylamide gels and immobilization on diisothiocyanate-glass beads in prepacked capillary columns for solid-phase microsequencing

A new method for immobilization of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to sequencing is described. It utilizes a simple apparatus that permits the simultaneous electroelution of proteins from gel slices and attachment to diisothiocyanate-activated glass beads prepacked in capillary tubes [S-P. Liang and R. A. Laursen, Anal. Biochem. 188, 366-373 (1990)]. Transfer/attachment yields of greater than 80% within 90 min were observed for several 125I-labeled proteins with a range of molecular weights using 0.2 M sodium phosphate (pH 8.9) buffer containing 0.1% SDS. The method has the advantage of high capacity, relative simplicity, and insensitivity to the presence of SDS and Coomassie blue stain. The highest transfer yields were obtained when proteins were run on gels which had been aged for at least 12 h. For 100- to 1000-pmol samples, the sequenceable amount of protein, including transfer, was generally 30-60%, with an average repetitive yield of 95%. Factors which influence sample recovery and sequencing yield are discussed.     

Validation of a liquid chromatographic method for the determination of ibuprofen in human plasma

A method based on LC-MS-MS is described for the determination of methyldopa in human plasma using dopa-phenyl-D3 as the internal standard. The method has a chromatographic run time of 5.5 min and was linear in the range of 20-5000 ng/ml. The limit of quantitation was 20 ng/ml, the intra-day precisions were 7.3, 5.4 and 4.3% and the intra-day accuracies were -8.0, -1.3 and -2.0% for 30, 600 and 3000 ng/ml, respectively. The inter-day precisions were 7.7, 0.5 and 0.7% and the inter-day accuracies were 0.2, -1.1 and -2.3%, respectively, for the above concentrations. This method was employed in a bioequivalence study of two tablet formulations of methyldopa.     

Protein patterns obtained from support-bound gels by combining the images at different stages of destaining

A more informative method for the visualization of proteins on thin-layer gels, based on combining the images of the gel at different stages of destaining, is presented. It is useful whenever important information seems to be lost after prolonged destaining. The method, which makes it unnecessary to run different loads in different channels, has been developed utilizing isoelectric focusing on polyester film-bound agarose gels. The strongly destained gel is superimposed on a negative image of the same gel made at an earlier phase of destaining, thus showing white spots on a gray background for minor components and dark bands in a white field surrounded by the grey background for the abundant ones. In general, the method may be applied to gel images obtained by different staining procedures.     

Silver staining of native and denatured eucaryotic DNA in agarose gels

A modified method of silver staining for native and denatured eucaryotic DNA in 1% agarose gel is described. This method is at least fivefold more sensitive than ethidium bromide staining, with a detection limit of 2.5 ng for total DNA. The calibration curve is linear within the range 5-30 ng of single-stranded and double-stranded DNA. This method is especially advantageous for electrophoretic assessment of DNA molecular weights.     

Reliable and specific high-performance liquid chromatographic method for simultaneous determination of loratadine and its metabolite in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the simultaneous determination of loratadine (L) and its metabolite, descarboethoxyloratadine (DCL), in human plasma. Following a two-step liquid-liquid extraction with toluene, the analytes were separated using a gradient mobile phase consisting of methanol-acetonitrile-phosphate buffer. The linearity for L and DCL was within the concentration range of 0.5-16 ng/ml. The coefficient of variation of intra- and inter-day assay was <8.3%, with accuracy ranging from 98.3 to 105.7%. The lower limit of quantification was 0.5 ng/ml for both L and DCL. This method has been demonstrated to be reliable, and is an improvement over existing methods due to its capability for determining L and DCL simultaneously in a single chromatographic run.     

Substrate-containing gel electrophoresis: sensitive detection of amylolytic, nucleolytic, and proteolytic enzymes

Electrophoresis of hydrolytic enzymes under nondenaturing conditions on acrylamide gels containing the appropriate high-molecular-weight substrates entrapped on the gel has been explored as a general method for sensitive enzyme resolution and detection. Under electrophoresis conditions of optimal enzyme activity, the enzymes may bind tightly to the fixed substrate and can only migrate in the electrophoretic field as the substrate is hydrolyzed. When the gels after electrophoresis in this “binding mode” are stained with substrate-detecting reagents, clear tracks of enzyme migration are observed, and the length of each track is a function of the amount of enzyme present in that track. Multiple forms of a given enzyme activity have not been and are not likely to be observed under these conditions. Under electrophoresis conditions of minimal (or suboptimal) enzyme activity, the enzymes do not bind to the fixed substrate and their mobility in the electrophoretic field does not appear to be significantly affected by the presence of substrate. After electrophoresis in this “nonbinding mode” the gels are incubated under conditions of optimal enzyme activity to allow substrate hydrolysis to take place before they are stained with substrate-detecting reagents, and active enzymes are detected as clear bands. Multiple forms of a given activity which were resolved during electrophoresis in the nonbinding mode are reflected by the presence of individual bands. The substrate-containing gel electrophoresis technique does not appear to be amenable to precise quantification of enzymes. By comparing the length of the clear tracks or the degree of staining of the activity bands for a range of enzyme concentrations, however, it is possible to establish the smallest amount of enzyme that can unequivocally be detected under a given set of conditions; from such studies we estimate that the sensitivity of detection with the substrate-containing gel electrophoresis technique can be orders of magnitude better than that obtained with other methods. The levels of detection observed in the work presented here were about 50 pg for α-amylase run on starch-containing gels, 1 pg to 1 ng for nucleases run on DNA- or RNA-containing gels, and 100 pg to 10 ng for 11 different pure and crude protease preparations run on gels containing heat-denatured bovine serum albumin.     

Simple and rapid determination of 1-deoxynojirimycin in mulberry leaves

A simple and rapid method for determining 1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor present in mulberry leaves (Morus alba and Morus bombysis), by high performance liquid chromatography coupled to an evaporative light scattering detector (ELSD) has been developed. DNJ was separated from extract of mulberry leaves on TSK gel Amide-80 column, which is a representative column for hydrophilic interaction chromatography. During post column detection, DNJ was detected by ELSD and concurrently identified by mass spectrometry. The detection limit was 100 ng. This method is sufficiently sensitive for determining DNJ in mulberry leaves and other related products.     

Hyaluronidase polymorphism detected by polyacrylamide gel electrophoresis. Application to hyaluronidases from bacteria, slime molds, bee and snake venoms, bovine testes, rat liver lysosomes, and human serum

A gel electrophoretic technique which allows detection of hyaluronidase activity in the gel has been devised. The principle is that the high-molecular-weight substrate, hyaluronic acid, is included in the gel, where it cannot move in the electrical field. After the run, the gel is incubated under conditions allowing the enzyme to degrade the substrate. Upon staining with "Stains-all" dye (Eastman Kodak Co., 2718), zones of hyaluronidase activity appear as pink bands in a blue background. The sensitivity limit is less than 3 fkat equivalent to 2.2 NF mU. The method is applicable to all types of hyaluronidases and chondroitinase ABC. It enabled to be shown that some hyaluronidases are polymorphic. This technique also made it possible to detect easily hyaluronidase activity in normal human serum. This analytical method represents a convenient step in the purification of hyaluronidase.     

High-performance liquid chromatographic-tandem mass spectrometric method for the determination of clemastine in human plasma

A highly sensitive high-performance liquid chromatographic-tandem mass spectrometric method (HPLC-MS-MS) has been developed to quantitate clemastine in human plasma for the purpose of pharmacokinetic studies. Sample preparation was carried out by liquid-liquid extraction using deuterated clemastine as an internal standard. Chromatographic separation used a C18 reversed phase polymer column giving an extremely fast total run time of 2 min. The method was validated and used for the bioequivalence study of clemastine tablets in healthy male volunteers (n=28). The lower limit of detection proved to be 0.01 ng/ml for clemastine.     

Simple high-performance liquid chromatographic method for determination of ketoconazole in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of ketoconazole in human plasma. The method entailed direct injection of the plasma sample after deproteinization using acetonitrile. The mobile phase comprised 0.05 M disodium hydrogen orthophosphate and acetonitrile (50:50, v/v) adjusted to pH 6. Analysis was run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The method is specific and sensitive with a quantification limit of approximately 60 ng/ml and a detection limit of 40 ng/ml at a signal-to-noise ratio of 3:1. Mean absolute recovery value was about 105%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 14%. The calibration curve was linear over a concentration range of 62.5–8000 ng/ml.     

Analytical techniques for cell fractions. XXII. Two-dimensional analysis of serum and tissue proteins: multiple gradient-slab gel electrophoresis

Two-dimensional electrophoresis of proteins and protein subunits, employing isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate in the second, yields the highest resolutions currently available. In this paper separations in the second dimension are considered (the so called DALT system). Methods for multiple-parallel casting of gradient gels in slab gel holders are described. The problem of electrical isolation of the ends of the slabs together with continuous cooling of both surfaces of the slab gel holder along their entire length has been achieved by running the gels in a horizontal direction in a three-compartment tank with the holders inserted in insulating septa. In the system described, 10 slabs are run simultaneously. This, however, is not the upper limit of the number of slabs which can be conveniently run in parallel.     

Simple high-performance liquid chromatographic method for the determination of ranitidine in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate–acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15–2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.