A vegetalizing inducing factor isolation and chemical properties

A vegetalizing factor which induces the formation of endodermal and mesodermal organs in amphibian gastrula ectoderm was purified from chicken embryos. Preparative sodium dodecyl sulfate polyacrylamide electrophoresis and gel permeation chromatography on Sephadex with different eluants were employed. In buffer containing 6 M urea the molecular weight of the factor was estimated to about 28 000–30 000. In buffer containing sodium dodecyl sulfate (SDS) the factor partially dissociates to smaller polypeptide chains. Because an equilibrium between molecules of different size is established, SDS-containing buffers are not suitable preparative purposes. In 50%–70% formic acid the factor completely dissociates into smaller peptide chains (M_r about 13 000–15 000). Furthermore, very little adsorption of the factor to the gel matrices or glass surfaces is observed in formic acid. The final purification can be achieved by high-performance gel permeation chromatography with glycerolpropyltreated silica gel as column packing and 50% formic acid as eluant.     

Preparative isolation of individual tRNA Phe by high performance liquid reversed-phase chromatography

A method of rapid preparative isolation of Phe-tRNA(Phe) (E. coli) of almost 100% purity (1800 pmoles per 1 OE260) is developed. In includes three successive chromatographies of total tRNA on C18-modified Lichrosphere Si-1000 in acetonitrile gradients. Other individual tRNAs can be isolated by the same approach.     

An improved flame ionization detector for high performance liquid chromatography

A flame ionization detector and associated transport device of high sensitivity for liquid chromatography are described. The system is based on the moving chain principle and employs a stainless steel belt of perforated structure that confers on it superior surface properties for collection and transport of column eluent. After evaporation of the solvent, the solute is converted to hydrocarbons in a stainless steel reactor of sandwich construction and is combusted in a hydrogen flame. The universality of the system is demonstrated with albumin and glucose, as well as with lipids. Sensitivity and reproducibility of the system are demonstrated also with a standard mixture of neutral lipids.     

Tetrahydrocurcumin in plasma and urine: quantitation by high performance liquid chromatography

Tetrahydrocurcumin (THC), one of the major metabolites of curcumin, exhibits many of the same physiologic and pharmacological activities as curcumin and in some systems may exert greater antioxidant activity than curcumin. However, evaluation of clinical efficacy is limited by lack of sensitive methods for quantifying intake/absorption in blood or urine. We have developed a sensitive high performance liquid chromatography (HPLC) analytical method for detection of THC in plasma and urine. The method involves extracting the THC from 0.2 mL samples with 95% ethyl acetate/5% methanol, and beta-17-estradiol acetate as an internal standard. Analysis with a reversed-phase C18 column and UV detection at 280 nm demonstrates linear performance from 0.050 to 6.0 microg/mL in plasma, and 0.060 to 6.0 microg/mL in urine. The coefficients of variation for intra- and inter-assays were each<8.6%. The average recovery of THC from plasma and urine was greater than 98.5%. These data demonstrate a rapid, sensitive and accurate method for HPLC quantification of THC in plasma and urine.     

Determination of methylputrescine oxidase by high performance liquid chromatography

N-Methyl-Δ¹-pyrrolinium chloride, the product of the title enzyme, was synthesized by methylation of aminobutyraldehyde diethylacetal followed by acidic cleavage. After purification to homogeneity, it was characterized by NMR and UV spectroscopy. The compound had an absorption maximum at 210 nm; previous data indicating a maximum at 267 nm were shown to arise from an impurity. An HPLC method for the assay of N-methylputrescine oxidase from plant material was developed based on the separation of N-methyl-Δ¹-pyrrolinium chloride on a cation exchange column and direct detection at 210 nm. The enzyme activity was measured in the protein fraction extracted from plant roots and treated by gel filtration on disposable PD 10 columns. A K_m value of 1.9 mM was determined for methylputrescine and the enzyme from tobacco roots. The enzyme activities from N. tabacum and Datura stramonium were compared.     

Automated and unbiased classification of chemical profiles from fungi using high performance liquid chromatography

In this paper we present a method for unbiased/unsupervised classification and identification of closely related fungi, using chemical analysis of secondary metabolite profiles created by HPLC with UV diode array detection. For two chromatographic data matrices a vector of locally aligned full spectral similarities is calculated along the retention time axis. The vector depicts the evaluating of the alikeness between two fungal extracts based upon eluted compounds and corresponding UV-absorbance spectra. For assessment of the chemotaxonomic grouping the vector is condensed to one similarity describing the overall degree of similarity between the profiles. Two sets of data were used in this study: One set was used in the method development and a second dataset used for method validation. First we developed a method for evaluating the secondary metabolite production from closely related Penicillium species. Then the algorithm was validated on fungal isolates belonging to the genus Alternaria. The results showed that the species may be segregated into taxa in full accordance with published taxonomy.     

Serum fatty acids analysis by high performance liquid chromatography, after enzymatic hydrolysis and isolation by Sep-Pak C18 minicolumn

A simple method of high performance liquid chromatography for the determination of fatty acid composition of free fatty acid, triglyceride, cholesterol ester and phospholipid in 0.4 ml of human serum is described. The procedure includes the enzymatic hydrolysis of serum lipoproteins, the isolation of fatty acids using Sep-Pak C18 minicolumn, the p-bromo-phenacylester formation and the high performance liquid chromatography. Thirteen fatty acids including the internal standard separated into 10 peaks within a 30-min run. The recovery rates of Sep-Pak treatment were satisfactory. The coefficients of variation were 2-27%. The present method showed a limit of detection of 0.02-4 nEq fatty acid.     

Analysis of protein-glutathione mixed disulfides by high performance liquid chromatography

After precipitation of proteins; serum, hepatocytes, or glutathione-derivatized bovine serum albumin, by perchloric acid, dithiotheritol was used to reduce glutathione-protein mixed disulfides in the ether-washed, resuspended pellet. Following neutralization and S-carboxymethylation of free sulfhydral groups in the acid soluble fraction by iodoacetic acid, 2,4-dinitrophenyl derivatives of released compounds were produced by addition of ethanolic fluorodinitrobenzene. The 2,4-dinitrophenyl derivative of S-carboxymethylglutathione was measured by high-performance liquid chromatography. The method was found to be reproducible and limited only by the sensitivity of the glutathione analysis (about 10 pmol/sample). Quantitation of protein-bound glutathione was shown to be indepedent of the ratio of bound to soluble glutathione as well as the protein concentration in the sample. This method was found to produce glutathione values identical to those measured after borohydride reduction without the problems of foaming, sample loss, and the need of continuous pH adjustment during reduction.     

Assay of bovine fibrinopeptides by high performance liquid chromatography.

A method for separating small amounts (<10^?5 mol) of bovine fibrinopeptides A and B employing high performance liquid chromatography has been developed. The limit of detectability of this method is about 10^?10 mol of fibrinopeptide. The separation was achieved within 20 min under reversed phase conditions using isocratic elution with aqueous buffer-acetonitrile solvent systems.     

高效液相法测定蜂蜜中甘油的含量

建立了以混合溶剂直接提取测定蜂蜜中甘油含量的方法.采用ZORBAX Carbohydrate a-nalysis(4.6 mm×250 mm 5-Micro)色谱柱,以乙腈/水(80:20,v/v)为流动相,示差检测器,使用乙腈/甲醇/水混合作为溶剂快速检测甘油.结果表明,应用此法的甘油浓度在10.0 mg/L～250...     

Analysis of bacterial menaquinone mixtures by high performance liquid chromatography

Menaquinone mixtures of microbial origin were separated according to the chain length and the degree of hydrogenation of the polyisoprenyl side-chain by reversephase partition high performance liquid chromatography. Menaquinones can be measured easily and sensitively by the chromatographic system described here.     

Purification of Clostridium perfringens enterotoxins by high performance liquid chromatography

Abstract Treatment of Saccharomyces cerevisiae a cells with α-factor partially inhibits mannosylation of the high M _r mannoproteins, although there is an increase in the total amount of these molecules present in the wall. They show a similar mobility in SDS-acrylamide gels to those from untreated mnn2 cells. No other significant effects on wall mannoproteins have been observed, except a decrease in the amount of the 29 kDa species.     

Analysis of apolipoproteins in high density lipoproteins by high performance liquid chromatography

A simple and rapid method for the analysis of apolipoproteins in high density lipoprotein (HDL) by high performance liquid chromatography (HPLC) has been developed (Kinoshita et al. (1983) J. Biochem. 94, 615-617). With this method, using a sodium phosphate buffer containing 0.1% sodium dodecyl sulfate (SDS) as an eluent, apolipoproteins can be analyzed from a very small amount of HDL fraction without delipidation using organic solvents. Separation profiles of apolipoproteins by this method were examined using several techniques. The elution pattern monitored by A280 can give precise quantitative as well as qualitative information about size-distribution of apolipoproteins, except for the apo C group. Moreover, separation of apo E from apo A-I was found to be improved by column elongation.     

Fractionation of fecal neutral steroids by high performance liquid chromatography

Fecal neutral steroids were fractionated by high performance liquid chromatography (HPLC) into three major fractions: 5 beta-H, 3-keto steroids; 5 beta-H, 3 beta-hydroxy steroids; and 5 alpha-H and delta 5-3 beta-hydroxy steroids. This separation was achieved in about 10 minutes, with greater than 97% recovery of standards in each fraction. Gas-liquid chromatographic quantitation of fecal steroids fractionated by either HPLC or thin-layer chromatography gave nearly identical results. A method using both C18 reverse phase and silica HPLC to purify radiolabeled sterols is also described.     

Analysis of aminophospholipid molecular species by high performance liquid chromatography

A new method is described for the separation of individual molecular species of the aminophospholipids, phosphatidylethanolamine and phosphatidylserine. Trinitrobenzene-sulfonic acid was used to derivatize both aminophospholipids and the derivatives were purified by thin-layer chromatography. A reversed-phase high performance liquid chromatography technique was developed to separate and quantify individual molecular species based upon ultraviolet detection of the attached chromophore. The retention times of the molecular species on the C18 reversed-phase column were longer with increasing carbon chain length and decreasing degree of unsaturation of fatty acyl chain. The overall procedure allowed a quantitative recovery of the aminophospholipid species. The lower limit of detection was about 10 pmol and a linear response was observed in the range of 0.1-10 nmol of phospholipid. Using this method, we were able to separate and quantify trinitrophenyl-phosphatidylethanolamine molecular species of both subclasses (diacyl and alkenyl) from human red blood cells and rat brains. Separation of species was confirmed by gas-liquid chromatographic analysis of the fatty acid content of each peak and by thermospray liquid chromatography-mass spectrometry. This new method provides a convenient and sensitive technique for studies of aminophospholipid molecular species composition. Furthermore, it appears to be a useful tool for the analysis of asymmetric distribution of these species in biological membranes.     

Compositional and molecular species analysis of phospholipids by high performance liquid chromatography coupled with chemical ionization mass spectrometry

High performance liquid chromatography (HPLC) was combined with chemical ionization mass spectrometry (CIMS) by the use of a moving-belt interface. The technique was employed for the analysis of naturally occurring phospholipids. Positive and negative ion mass spectra of various phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin were obtained in the chemical ionization mode with ammonia or methane as the reagent gas. Specific ions for individual phospholipid "bases" were identified. These ions were used in specific ion monitoring of the phospholipids during HPLC-CIMS. CIMS of each phospholipid also provided extensive information on the molecular species of the individual class of phospholipids. Relative abundance of different molecular species of each phospholipid as determined by CIMS agreed well with the results obtained by gas-liquid chromatography. Rat brain phospholipids were analyzed by HPLC-CIMS in about 15 minutes. Routinely, about 5 micrograms of individual phospholipid was analyzed by HPLC-CIMS, however, with specific ion monitoring the method provides a detection capability at the subnanogram level.