Isolation and identification of rutin as the major mutagen of red wine

The polyphenolics of a red wine were concentrated by salt-induced phase separation into acetone-alcohol and fractionated by Sephadex LH-20 and multi-layer counter-current chromatography. The mutagenicity of each fraction was evaluated by the Salmonella mutagenesis assay. The mutagen of red wine required activation by both rat-liver microsomal enzymes and human-fecal enzymes (fecalase). The mutagenic component of red wine was purified to homogeneity by reverse-phase high-performance liquid chromatography (RPHPLC) on Lichrosorb C18 and was identified as rutin by UV spectrometry, co-chromatography with authentic standard on RPHPLC and gas-liquid chromatography/mass spectrometry.     

Variables in the high-pressure cation-exchange chromatography of proteins

The use of cation-exchange high-pressure liquid chromatography for the separation of proteins has been investigated. Several factors, including solvent composition, pH, flow rate, and temperature, were examined for their effects on the resolution of protein standards (insulin, β-lactoglobulin, and carbonic anhydrase B; molecular weight range, 6000 to 30,000 and pI range, 5.3 to 6.5). An initial comparison was made of the recovery of these proteins from three commercially available columns (Whatman Partisil SCX, Separation Industry CM silica, and MCB Reagents Lichrosorb KAT). In general, under the conditions employed, the SCX column gave the highest recovery of applied protein. Based on this recovery data, the Partisil SCX column was chosen for subsequent examination of chromatographic parameters that would optimize protein resolution. An increase in temperature decreased retention and resolution but increased recovery, with some proteins being affected more than others. A decrease in pH in the final eluant or an increase in pH in the initial eluant caused an increase in retention times. For some proteins, the decrease in pH resulted in a greater total recovery of protein. This information has been applied to the purification by cation-exchange high-pressure liquid chromatography of transforming growth factors from a human tumor cell line.     

Isolation of secondary metabolites of Aspergillus ochraceus by HPLC

A method is described for the isolation and purification of ochratoxin A, ochratoxin B, ochratoxin ß mellein, 4-hydroxymellein and penicillic acid produced byAspergillus ochraceus in a synthetic liquid medium. Ochratoxin α, which was not found in the culture medium, was obtained by acid hydrolysis of ochratoxin A. A high pressure liquid Chromatograph equipped with Lichrosorb 100 and Lichrosorb RP-18 columns and UV and/or Refractive Index detectors was used.     

Alkaline extraction and reverse-phase high-performance liquid chromatography of adenine and pyridine nucleotides in human platelets

The levels of adenine (ATP, ADP, AMP) and pyridine (NAD, NADH) nucleotides in human platelets have been measured by a simple and reproducible method. A rapid alkaline extraction allows a complete recovery of the compounds concerned. The metabolic ATP and ADP in the cytosolic fraction, the amount released upon thrombin stimulation, and the ADP bound to F-actin have also been evaluated. Analysis was performed by reverse-phase, isocratic high-performance liquid chromatography on a 5-microns Lichrosorb RP-18 column with uv detection at 254 nm.     

Characterization of isoprenoid involved in the post-translational modification of mammalian cell proteins

Mammalian cell proteins, modified post-translationally by derivatives of [3H]mevalonic acid, were subjected to methylation and sulfonium salt cleavage reactions previously used to release isoprenoids from cysteine residues in yeast peptides. The labeled isoprenoid extracted into chloroform comigrated with farnesol through a series of chromatography steps including Sep-Pak C-18 fractionation, size exclusion on Bio-Beads, and reverse-phase chromatography. Further resolution of the material by normal phase liquid chromatography and thin layer chromatography demonstrated the presence of farnesol, nerolidol, and other unidentified hydrophobic derivatives. Similar products were generated when S-farnesyl cysteine was subjected to the methylation and cleavage procedures. These preliminary findings suggest that farnesylation of cysteine residues accounts for the well documented incorporation of mevalonic acid into mammalian cell proteins.     

Urinary protein profiling by high-performance gel permeation chromatography

We describe a new application of high-performance aqueous gel permeation chromatography for the analysis of human proteinuria. Separations of urinary proteins from normal subjects and patients with renal impairment were performed with TSK G 3000 SW columns. The effects of pH and icnic strength of the eluent on the separation of urinary proteins were investigated. Albumins were selectively separated from urine by affinity chromatography on Blue Sepharose CL-6B. According to the results of clinical investigations, urinary protein pattern derived from gel permeation chromatography revealed a good prediction of the site of renal involvement. Predominant excretion of proteins with lower molecular weight than albumin correlated with tubular damage. Albumin and higher molecular weight protein patterns were associated with glomerular disease. Absorbance measurements of the eluent at 280 nm were used for quantitative determination of total urinary protein. Gel permeation chromatography was compared to sodium dodecyl sulfate—polyacrylamide gel electrophoresis and the resulting protein patterns are in good agreement.     

Proteomic profiling of nonenzymatically glycated proteins in human plasma and erythrocyte membranes

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this work, we report the first proteomics-based characterization of nonenzymatically glycated proteins in human plasma and erythrocyte membranes from individuals with normal glucose tolerance, impaired glucose tolerance, and type 2 diabetes mellitus. Phenylboronate affinity chromatography was used to enrich glycated proteins and glycated tryptic peptides from both human plasma and erythrocyte membranes. The enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation-tandem mass spectrometry, resulting in the confident identification of 76 and 31 proteins from human plasma and erythrocyte membranes, respectively. Although most of the glycated proteins could be identified in samples from individuals with normal glucose tolerance, slightly higher numbers of glycated proteins and more glycation sites were identified in samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus.     

Cytochalasin B: preparation, analysis in tissue extracts, and pharmacokinetics after intraperitoneal bolus administration in mice

Cytochalasin B (CB) was prepared by methanol extraction of dehydrated mold (Drechslera dematioidea) matte, reverse-phase C18 silica gel batch adsorption, selective elution with 1:1 (v/v) hexane:tetrahydrofuran (THF), crystallization, preparative TLC, and recrystallization. Unit gravity silica gel normal phase chromatography afforded additional CB. Yield per liter of medium was 300 mg of CB greater than 95% pure by NMR, HPLC (60:40 hexane:THF, Lichrosorb Si60 silica gel, 230 nm), and TLC. CB added exogenously to mouse organs at 1 and 5 micrograms/organ was recovered 70 to 100% by methanol extraction, adsorption to C18 silica gel Sep-Pak cartridges, elution with ethyl acetate, and analysis by TLC and/or HPLC. Limiting sensitivity (micrograms/extract) was 0.5 TLC; 1.0 HPLC. Quantitative extraction was confirmed with 3H-labeled CB. CB ip in mice at 50 mg/kg (LD10) distributed rapidly into liver, renal fat, kidney, intestines, mesentery, pancreas, spleen, and blood cells and was cleared from all but liver within 24 h. CB was below detectable levels in thymus, lymph nodes, heart, brain, bone marrow, and lungs. Cytochalasin A is fixed to tissues and not extractable. This work affords a source of CB in quantities permitting in vivo study, provides methods for extraction and analysis, and reveals the pharmacokinetics of ip bolus CB.     

Separation and quantification of chicken and bovine growth plate cartilage matrix vesicle lipids by high-performance liquid chromatography using evaporative light scattering detection

Matrix vesicles (MV) are lipid bilayer-enclosed nanoscale structures that initiate extracellular mineral formation in most vertebrate species. Little attention has been given to differences between species in membrane lipid composition or to how new mineral is formed in MV. To explore more precisely the lipids of MV isolated from avian and bovine species, we developed a new high-performance liquid chromatography (HPLC) method used in combination with evaporative light scattering detection (ELSD) to quantify their lipid composition. HPLC analyses were performed on a Lichrosorb silica column using separate binary gradient elution systems for analyzing polar and nonpolar lipids. Standard mixtures of both phospholipids and nonpolar lipids were used to prepare calibration curves for each lipid, which were analyzed mathematically by polynomial regression for accurate quantitation. This new methodology provides high-resolution separations and quantitation of both the polar and the nonpolar lipids typically present in biological membranes, including lyso- (monoacyl-) phospholipids. We have applied this method to quantitate the phospholipid and nonpolar lipid composition of MV isolated from chicken and bovine growth plate cartilage. We also compared the ability of these MV to induce mineral formation. While the ability to induce mineralization and the lipid composition were generally similar, some significant differences between MV from these two disparate species were seen.     

Characterization of a fetal calf serum-derived molecule reactive with human natural antibodies: its occurrence in tissue culture-grown type C RNA viruses.

By means of a sensitive radioimmunoprecipitation (RIP) assay, simian sarcoma virus-simian sarcoma-associated virus (SSV-SSAV), purified from culture fluids of infected normal rat kidney (NRK) cells, was shown to acquire a surface antigen from serum used in the tissue culture medium. This antigen, which was acquired when serum from either fetal calf, horse, swine, rabbit, or chicken origin was used, accounted for a substantial portion (but not all) of the total precipitating activity exhibited by natural human antibodies for membrane-associated antigens of these viruses. By 1) alcohol precipitation, concanavalin A chromatography, and Sephadex G-150 filtration of fetal calf serum (FCS) proteins or 2) chromatography of serum proteins over a human IgG-containing immunoaffinity column, a glycoprotein of approximately 55,000 daltons has been identified which is a minor constituent of FCS (less than 0.1% of total protein) and has the antigenic capacity of whole FCS.     

In-depth analysis of the human CSF proteome using protein prefractionation

The identification of disease markers in human body fluids requires an extensive and thorough analysis of its protein constituents. In the present study, we have extended our analysis of the human cerebrospinal fluid (CSF) proteome using protein prefractional followed by shotgun mass spectrometry. After the removal of abundant protein components from the mixture with the help of immunodepletion affinity chromatography, we used either anion exchange chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to further subfractionate the proteins present in CSFs. Each protein subfraction was enzyme digested and analyzed by tandem mass spectrometry and the resulting data evaluated using the Spectrum Mill software. Different subfractionation methods resulted in the identification of a grant total of 259 proteins in CSF from a patient with normal pressure hydrocephalus. The greatest number of protein, 240 in total, were identified after prefractionating the CSF proteins by immunodepletion and SDS-PAGE. Immuno-depletion combined with anion exchange fractionation resulted in 112 proteins and 74 proteins were found when only immunodepletion of the CSF samples was carried out. All methods used showed a significant increase in the number of identified proteins as compared with nondepleted and unfractionated CSF sample analysis, which yielded only 38 protein identifications. The present work establishes a platform for future studies aimed at a detailed comparative proteome analysis of CSFs from different groups of patients suffering from various psychiatric and neurological disorders.     

Combination of abundant protein depletion and multi-lectin affinity chromatography (M-LAC) for plasma protein biomarker discovery

We report on the development of a robust and relatively high-throughput method for in-depth proteomic analysis of human plasma suitable for biomarker discovery. The method consists of depletion of albumin and IgG and multi-lectin affinity chromatography (M-LAC), followed by nanoLC-MS/MS analysis of digested proteins and label-free comparative quantitation of proteins. The performance of the method is monitored by multiple quality control points to ensure reproducibility of the analysis. The method identifies proteins that are reported to be present in normal plasma at concentrations of 10-100 ng/mL and that may be of particular interest when studying a variety of disease conditions. Numerous tissue leakage proteins of potentially even lower concentrations are also identified. When the method was used in a study to identify potential biomarkers of psoriasis, the differential abundance of proteins present at low mug/mL level was quantitated and later verified by ELISA measurements.     

Immobilization of L-glyceryl phosphorylcholine: isolation of phosphorylcholine-binding proteins from seminal plasma

The preparation of an affinity sorbent containing immobilized L-glyceryl phosphorylcholine for affinity chromatography of phosphorylcholine-binding proteins from seminal plasma is described. The ligand was coupled either after its maleinylation to poly(acrylamide-allyl amine) copolymer or directly to divinyl sulfone-activated Sepharose. The prepared phosphorylcholine derivative coupled to Sepharose was used for affinity chromatography of phosphorylcholine-binding proteins from bull and boar seminal plasma. Adsorbed proteins were specifically eluted with phosphorylcholine solution. Isolated phosphorylcholine-binding proteins were characterized by SDS electrophoresis and HPLC with reversed phase. Composition of the boar phosphorylcholine-binding fraction obtained by affinity chromatography on immobilized L-glyceryl phosphorylcholine was compared with that eluted from immobilized heparin by the phosphorylcholine solution. No phosphorylcholine-binding proteins were found in human seminal plasma.     

Antigenic determinants of blood serum proteins detected in the structure of hepatitis B surface antigen by the method of affinity chromatography]

Tropism of the antigen of hepatitis B to the antibodies against normal serum proteins was revealed by the method of affine chromatography; this pointed to the possibility of the presence in the HBs-antigen structure of some antigenic determinants of the serum proteins or to the association of serum proteins with the HBs-antigen. A change of the isoelectric spectrum of the HBs-antigen and its tropism in affine chromatography to the antibodies against serum proteins possibly depended both on the nonhomogeneity of the antigenic determinants included into the composition of the antigen, and on the presence of the HBs-antigen--HBs-antibody or HBs-antigen--serum proteins complexes.     

Effects of cocultivation with transformed cells on surface proteins of normal cells

Virally transformed fibroblasts do not have on their surface a major protein (large external transformation-sensitive, LETS) which is present in normal cells. Cocultivation of the transformation cells with normal cells whose surface proteins have been prelabelled induces an accelerated release of the LETS protein from the normal cells. We have investigated various conditions which affect this phenomenon. Our results show that alteration of cell surface proteins by cocultivation with the transformed cells is time and dose-dependent and requires cell contact. Serum was depleted at least 99% of plasminogen by affinity chromatography and used in the cocultivation experiments. It was found that activation of plasminogen was not required for the accelerated turnover of the LETS protein. Other diffusible proteases are also unlikely to be involved. The possibility that transformed cells have a membrane bound activity is discussed. The role of plasminogen activation was also tested for its relevance in transformation related proteolysis, growth and morphology of cells.     

Effects of cocultivation with transformed cells on surface proteins of normal cells.

Virally transformed fibroblasts do not have on their surface a major protein (large external transformation-sensitive, LETS) which is present in normal cells. Cocultivation of the transformed cells with normal cells whose surface proteins have been prelabelled induces an accelerated release of the LETS protein from the normal cells. We have investigated various conditions which affect this phenomenon. Our results show that alteration of cell surface proteins by cocultivation with the transformed cells is time and dose-dependent and requires cell contact. Serum was depleted at least 99% of plasminogen by affinity chromatography and used in the cocultivation experiments. It was found that activation of plasminogen was not required for the accelerated turnover of the LETS protein. Other diffusible proteases are also unlikely to be involved. The possibility that transformed cells have a membrane bound activity is discussed. The role of plasminogen activation was also tested for its relevance in transformation related proteolysis, growth and morphology of cells.     

Immunospecificity of non-histone chromosomal proteins in 89Sr-induced osteogenic sarcoma (mouse).

Antibodies against non-histone chromosomal proteins for 89Sr-induced osteogenic sarcoma (mouse) were prepared by immunization of rabbits. The immunoreactivity of this antigen was then compared with those of non-histone chromosomal proteins from Ehrlich ascites tumor, normal mouse liver, and calf thymus by the method of quantitative microcomplement fixation. The non-histone chromosomal proteins of 98Sr-induced osteogenic sarcoma, fractionated by hydroxylapatite chromatography, exhibited significant affinity for the antibodies. Similar proteins from Ehrlich ascites tumor, normal mouse liver, or calf thymus were virtually inactive, indicating the tissue-specificity of 89Sr-induced osteogenic sarcoma proteins.     

包涵体蛋白的层析复性技术研究进展

包涵体蛋白的复性是生物工程下游技术中的一个重要难题。层析法用于蛋白质复性是一种较新的、适用于大多数蛋白的方法。其原理是将层析技术应用于蛋白质复性和纯化,使变性蛋白质在层析柱上重折叠为正确的空间构象,在洗脱的同时实现部分纯化。本文详细介绍了蛋白质在5种层析柱上的复性方法、原理、应用及研究的新进展,为层析法对蛋白质复性的进一步应用提供依据。