Cloud point extraction and preconcentration for the gas chromatography of phenothiazine tranquilizers in spiked human serum.

Cloud point extraction was successfully applied to the preconcentration of phenothiazine derivatives, such as pericyazine (PC), chlorpromazine (CP) and fluphenazine (FUL), for gas chromatography (GC). Phenothiazine derivatives were separated from surfactants by passing the surfactant-rich phase through a cation exchange column after cloud point extraction, permitting the determination of the phenothiazine derivatives extracted in the surfactant-rich phase by GC. The optimal condition for the cloud point extraction of phenothiazine derivatives was also investigated using Triton X-100, Triton X-114, and PONPE10. Triton X-114 provided the most efficient recovery of phenothiazine derivatives among the surfactants used. The addition of sodium chloride and excess ammonia to the sample solution resulted in a decrement of the recovery of the phenothiazine derivatives. The proposed method was applied to the determination of phenothiazine derivatives in spiked human serum by GC. The recoveries of PC, CP, and FUL in spiked human serum were 95.1%, 87.1%, and 84.7%, respectively.     

Sensitive determination of phenothiazines in pharmaceutical preparation and biological fluid by flow injection chemiluminescence method using luminol-KMnO4 system

A flow injection chemiluminescence method was described for the determination of four phenothiazine drugs, namely, chlorpromazine hydrochloride, perphenazine hydrochloride, fluphenazine hydrochloride and thioridazine hydrochloride. Strong Chemiluminescence (CL) signal was produced when above-mentioned drug was injected into the mixed stream of luminol with KMnO₄. The linear ranges of the method were 0.0020-1.0 μg/mL chlorpromazine hydrochloride, 0.0040-3.0 μg/mL perphenazine hydrochloride, 0.0020-5.0 μg/mL fluphenazine hydrochloride and 0.0050-1.0 μg/mL thioridazine hydrochloride. The detection limits were 0.4 ng/mL chlorpromazine hydrochloride, 0.7 ng/mL perphenazine hydrochloride, 2 ng/mL fluphenazine hydrochloride and 0.7 ng/mL thioridazine hydrochloride. The proposed method was applied to the determination of chlorpromazine hydrochloride in injections and in mental patient's urine samples and the satisfactory results were achieved. The possible CL reaction mechanism was also discussed briefly.     

Photobinding of 8-methoxypsoralen, 4,6,4'-trimethylangelicin and chlorpromazine to Wistar rat epidermal biomacromolecules in vivo.

Photoinduced binding of drugs to endogenous biomacromolecules may cause both toxic and therapeutic effects. For example, photobinding of certain phenothiazines to biomolecules possibly underlies their phototoxic and photoallergic potential, whereas photobinding of furocoumarins to epidermal DNA is held responsible for their advantageous effects in the photochemotherapy of psoriasis. Usually, the in vitro photobinding of drugs is investigated. However, under in vivo conditions, the metabolism and distribution of the drug and the light absorption by endogenous compounds will significantly affect the photobinding of drugs to biomolecules. Therefore, in the present study, the photobinding of 8-methoxypsoralen (8-MOP), 4,6,4'-trimethylangelicin (TMA) (two therapeutically used furocoumarins) and chlorpromazine (CPZ) (a member of the phenothiazines) was investigated in vivo. The compounds were applied topically on the shaven skin of Wistar rats; one group was exposed to UVA and the other was kept in a dimly lit environment. Immediately, and at certain time intervals after UVA exposure, members of the two groups were sacrificed. By separating epidermal lipids, DNA/RNA and proteins by a selective extraction method, irreversible binding of 8-MOP, TMA or CPZ to each of these biomacromolecules was determined. In contrast with in vitro experiments, photobinding of CPZ to epidermal DNA/RNA was not found in vivo; apparently the bioavailability in the nucleus is very low. Compared with TMA, 8-MOP was observed to bind more extensively to epidermal DNA/RNA (again in contrast with findings from in vitro experiments) and proteins, but less extensively to lipids. The rates of removal of photobound 8-MOP and TMA were comparable. Photobound CPZ was more slowly removed from epidermal proteins and lipids than the furocoumarins. The observed in vivo photobinding is discussed with respect to the UVA-induced (side) effects of these drugs.     

Two-dimensional electrophoresis of membrane proteins

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.     

Proteomic study of the peripheral proteins from thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803

Thylakoid membranes of cyanobacteria and plants contain enzymes that function in diverse metabolic reactions. Many of these enzymes and regulatory proteins are associated with the membranes as peripheral proteins. To identify these proteins, we separated and identified the peripheral proteins of thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Trichloroacetic acid (TCA)-acetone extraction was used to enrich samples with peripheral proteins and to remove integral membrane proteins. The proteins were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting. More than 200 proteins were detected on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel that was stained with colloidal Coomassie blue. We analyzed 116 spots by peptide mass fingerprinting and identified 78 spots that were derived from 51 genes. Some proteins were found in multiple spots, indicating differential modifications resulting in charge differences. Therefore, a significant fraction of the peripheral proteins in thylakoid membranes is modified post-translationally. In our analysis, products of 17 hypothetical genes could be identified in the peripheral protein fraction. Therefore, proteomic analysis is a powerful tool to identify location of the products of hypothetical genes and to characterize complexity in gene expression due to post-translational modifications.     

Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry

A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved in the range 2-10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 +/- 4 pg/0.0044 s was obtained for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma.     

High yield electroblotting onto polyvinylidene difluoride membranes from polyacrylamide gels.

Optimal conditions of electroblotting that led to high protein recovery on polyvinylidene difluoride (PVDF) membranes were determined for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS concentrations in the gel and transfer buffer were found to be the most important factors affecting the amount of protein recovered on the PVDF membrane. The largest loss occurred during the first 10-30 min of transfer due to the relatively high initial SDS concentration in the gel. During this initial stage of transfer, most of the protein passed through the primary membrane and was partially retained on secondary and tertiary membranes. The value of presoaking gels prior to transfer to reduce the amount of SDS was evaluated by quantitating free SDS densitometrically and by correlating the reduced SDS concentration with increased electroblotting efficiency from presoaked gels. Transfer time was evaluated and no "overtransfer" was found even after very long transfer times. These results clearly indicate that proteins electroblotted onto PVDF membranes were tightly bound and could not be released by extending the transfer time. The effects of methanol and SDS concentrations on protein adsorption from solution to PVDF were also determined quantitatively. The results of this study strongly suggest that proteins fully saturated with SDS cannot bind efficiently to PVDF membranes. Since SDS is necessary for high protein mobility, the challenge in efficient electroblotting is to maintain an optimal SDS concentration which is high enough to permit effective removal from the gel and low enough to permit effective binding to the PVDF membrane. For 1.5 mm thick gels containing 0.2% SDS, presoaking the gel for 15-20 min in transfer buffer with 10% methanol prior to electroblotting provided the best recovery on the primary membrane.     

Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry

A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved in the range 2– 10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 ± 4 pg/0.0044 s was obtained for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma. Received: 28 June 1999 / Revised: 14 September 1999 / Accepted: 16 September 1999     

Identification of peroxisomal membrane proteins of Saccharomyces cerevisiae by mass spectrometry

The identification of peroxisomal membrane proteins is very important to understand the import mechanisms of substrates and proteins into these organelles and the pathogenesis of human peroxisomal disorders like the Zellweger Syndrom. Peroxisomal membrane proteins were identified after separation by gel electrophoresis, tryptic digestion and mass spectrometric analysis. Using matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), it was possible to identify 45 proteins of isolated yeast peroxisomal membranes.     

Rapid detection of proteins by enzyme-linked immunofiltration assay after transfer onto nitrocellulose membranes.

Enzyme-linked immunofiltration assay (ELIFA) for labeling transferred proteins is an interesting and powerful technique for the rapid specific detection (15 min) of proteins immobilized on nitrocellulose or nylon membranes (0.20 and 0.45 micron). ELIFA does not require fastidious handling of the membranes. Saturation, specific labeling and washing procedures are achieved by filtration, controlled by a monitoring unit which regulates the flow rate and ensures excellent specificity, repetition and reproducibility. The recycling by closed circuit or by repetitive inversion of the flow direction offers the advantage of reducing the volumes of expensive reagents while simultaneously increasing the sensitivity of the technique. The detection limit is at least as low as 1-5 ng using directly or indirectly enzymatically labelled probes. ELIFA may be extended to the identification of glycoproteins using specific ligands such as lectins or to the immunocapture of an antigen using specific antibodies immobilized on an activated membrane. ELIFA complements fast separation, by e.g., isoelectric focusing, polyacrylamide gel electrophoresis, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis and accelerated electrotransfer to membranes with rapid detection reducing the total time for separation transfer and detection to less than 2 h.     

Analysis of transmembrane proteins from eukaryotic cells

The topography and properties of plasma membrane proteins from mouse L-929 cells are studied by comparing their availability for enzymatic labeling on the external and internal surfaces of the membrane. In order to study the internal surface, phagolysosomes are prepared from cells after they ingest latex particles. The plasma membrane surrounding these seem to have an "inside-out" orientation. The sugars of the membrane glycoproteins in intact phagolysosomes are not available for interaction with lectins or available for periodate-borotritide labeling. A comparison of the lectin-binding proteins labeled by lactoperoxidase-catalyzed iodination on the external cell surface with those labeled on the internal cell surface suggests that a variety of plasma membrane glycoproteins span the lipid bilayer. Using two-dimensional gel electrophoresis it has been shown that selected proteins are labeled at both the internal and external faces of the plasma membrane. Analysis of the 2-D gel electrophoregrams reveals that there are two distinct prominent proteins at 60,000 and 100,000 daltons which are enzymatically iodinated from both sides of the membrane. The partial hydrolysis of the 100,000 dalton protein reveals that different peptides are iodinated when the iodination is performed on intact cells or on the phagolysosomes. These proteins are extensively phosphorylated in cells incubated with inorganic 32P. We conclude that the phagolysosome is probably oriented in an "inside-out" configuration and that this membrane preparation can be used to study the topographic organization of membrane proteins. The use of oriented membranes, selective labeling of proteins, and affinity separation of proteins in combination with gel electrophoresis to define the position and properties of proteins is discussed.     

Direct N-terminal sequence analysis of rat liver plasma membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis

Nine previously uncharacterized membrane glycoproteins from normal rat liver have been analyzed by amino acid sequencing from two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after transblotting to Immobilon-P membranes. Three of these components show altered levels of expression in liver tumors. A single electroblotted polyacrylamide gel yielded sufficient quantities of these glycoproteins for amino acid sequencing and the N-terminal structure could be determined for four of them. The remaining five glycoproteins of interest were not sequenceable in this manner, presumably because they had blocked N-termini. Prior to electrophoresis, two enrichment methods were applied to the crude liver membrane preparations: affinity chromatography with concanavalin A to isolate the plasma membrane glycoproteins and then fast protein liquid chromatography on Superose 12 to obtain components having a specific range of molecular weights. These materials were next subjected to 2-D PAGE using pH 4-6 carrier ampholytes in the first dimension and 7.5% sodium dodecyl sulfate gels in the second. The proteins were then electroblotted to Immobilon-P membranes and located by staining with Coomassie Brilliant Blue R-250. Our results demonstrate that N-terminal sequencing (gas-phase) can be achieved on polypeptides obtained from approximately 250 micrograms of total glycoproteins applied to a single 2-D gel.     

Separation and identification of photosynthetic antenna membrane proteins by high- performance liquid chromatography electrospray ionization mass spectrometry

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes. Nevertheless, structural studies on this part of the proteome are limited. The present review attempts to cover the vast array of methods that have appeared in the last few years for separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts, a good model for setting up analytical methods suitable for membrane proteins. The two major methods for the separation of thylakoid membrane proteins are gel electrophoresis and liquid chromatography. Isoelectric focusing in a first dimension followed by denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a second dimension is an effective way to resolve large numbers of soluble and peripheral membrane proteins. However, it is not applicable for isolation of native protein complexes or for the separation of highly hydrophobic membrane proteins. High-performance liquid chromatography (HPLC), on the other hand, is highly suitable for any type of membrane protein separation due to its compatibility with detergents that are necessary to keep the hydrophobic proteins in solution. With regard to the identification of the separated proteins, several methods are available, including immunological and mass spectrometric methods. Besides immunological identification, peptide mass fingerprinting, peptide fragment fingerprinting or intact molecular mass determination by electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been shown to be very sensitive and effective. In particular, identification of proteins by their intact molecular mass is advantageous for the investigation of numerous biological problems, because it is rapid and reflects the full sequence of the protein and all its posttranslational modifications. However, intact molecular mass determinations of gel-separated membrane proteins are hampered due to the difficulties in extracting the hydrophobic proteins from the gel, whereas HPLC on-line interfaced with ESI-MS enables the rapid and accurate determination of intact molecular masses and consequently an unequivocal protein identification. This strategy can be viewed as a multidimensional separation technique distinguishing between hydrophobicity in the first dimension and between different mass-to-charge ratios in the second dimension, allowing the separation and identification even of isomeric forms.     

亲和膜色谱法纯化人血浆纤溶酶原

首次以尼龙膜为基质 ,分别采用三氯三嗪和 1 ,4 丁二醇二缩水甘油醚活化法 ,制备了以L 赖氨酸为配基的亲和膜。首次使用亲和膜色谱法成功地从人血浆中快速纯化出了高纯度的纤溶酶原 ,电泳分析显示一个主要的谱带。为规模化快速纯化临床用高纯度纤溶酶原提供了新方法。     

IRREVERSIBLE PHOTOBINDING TO SKIN CONSTITUENTS AFTER SYSTEMIC ADMINISTRATION OF CHLORPROMAZINE TO WISTAR RATS

From in vitro experiments it is known that chlorpromazine binds to protein and DNA/ RNA upon UV-irradiation. In the present study the possible photobinding of chlorpromazine (or its metabolites) in vivo was examined. Tritium labeled drug was administered intraperitoneally to female albino Wistar rats after which they were irradiated with light with maximum intensity at 310, 370 or 420 nm. After homogenization, unbound radioactivity in tissue of several organs was removed by dialysis. In the ears, eyes and skin of the back irreversibly bound radioactivity could be detected after irradiation with 310- and 370- but not with 420 nm light. Binding in the skin of the back after UVA irradiation was examined in more detail by separating epidermal lipids, DNA/RNA and proteins by a selective extraction/precipitation method. Radioactivity appeared to be bound to lipids and proteins but not to DNA/RNA.     

Separation and identification of the light harvesting proteins contained in grana and stroma thylakoid membrane fractions

This paper presents the results of a study performed to develop a rapid and straightforward method to resolve and simultaneously identify the light-harvesting proteins of photosystem I (LHCI) and photosystem II (LHCII) present in the grana and stroma of the thylakoid membranes of higher plants. These hydrophobic proteins are embedded in the phospholipid membrane, and their extraction usually requires detergent and time consuming manipulations that may introduce artifacts. The method presented here makes use of digitonin, a detergent which causes rapid (within less than 3 min) cleavage of the thylakoid membrane into two subfractions: appressed (grana) and non-appressed (stroma) membranes, the former enriched in photosystem II and the latter containing mainly photosystem I. From these two fractions identification of the protein components was performed by separating them by reversed-phase high-performance liquid chromatography (RP-HPLC) and determining the intact molecular mass by electrospray ionization mass spectrometry (ESI-MS). By this strategy the ion suppression during ESI-MS that normally occurs in the presence of membrane phospholipids was avoided, since RP-HPLC removed most phospholipids from the analytes. Consequently, high quality mass spectra were extracted from the reconstructed ion chromatograms. The specific cleavage of thylakoid membranes by digitonin, as well as the rapid identification and quantification of the antenna composition of the two complexes facilitate future studies of the lateral migration of the chlorophyll-protein complexes along thylakoid membranes, which is well known to be induced by high intensity light or other environmental stresses. Such investigations could not be performed by sodium dodecylsulfate-polyacrylamide gel electrophoresis because of insufficient resolution of the proteins having molecular masses between 22,000 and 25,000.     

Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin

The release of neurotransmitter is regulated in the processes of membrane docking and membrane fusion between synaptic vesicles and presynaptic plasma membranes. Synaptic vesicles contain a diverse set of proteins that participate in these processes. Small GTP-binding proteins exist in the synaptic vesicles and are suggested to play roles for the regulation of neurotransmitter release. We have examined a possible role of GTP-binding proteins in the regulation of protein phosphorylation in the synaptic vesicles. GTPgammaS stimulated the phosphorylation of 46 kDa protein (p46) with pI value of 5.0-5.2, but GDPbetaS did not. The p46 was identified as protein interacting with C-kinase 1 (PICK-1) by MALDI-TOF mass spectroscopy analysis, and anti-PICK-1 antibody recognized the p46 spot on 2-dimensional gel electrophoresis. Rab guanine nucleotide dissociation inhibitor (RabGDI), which dissociates Rab proteins from SVs, did not affect phosphorylation of p46. Ca(2+)/calmodulin (CaM), which causes the small GTP-binding proteins like Rab3A and RalA to dissociate from the membranes and stimulates CaM-dependent protein kinase(s) and phosphatase, strongly stimulate the phosphorylation of p46 in the presence of cyclosporin A and cyclophylin. However, RhoGDI, which dissociates Rho proteins from membranes, reduced the phosphorylation of p46 to the extent of about 50%. These results support that p46 was PICK-1, and its phosphorylation was stimulated by GTP and Ca(2+)/CaM directly or indirectly through GTP-binding protein(s) and Ca(2+)/CaM effector protein(s). The phosphorylation of p46 (PICK-1) by GTP and Ca(2+)/CaM may be important for the regulation of transporters and neurosecretion.     

Isolation of Escherichia coli inner membranes by metal affinity two-phase partitioning

As reduction of sample complexity is a central issue in membrane proteomic research, the need for new pre-fractionation methods is significant. Here we present a method for fast and efficient enrichment of Escherichia coli inner membranes expressing a His-tagged integral membrane L-fucose-proton symporter (FucP). An enriched inner membrane fraction was obtained from a crude membrane mixture using affinity two-phase partitioning in combination with nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. Due to interaction between the beads and FucP, inner membranes were selectively partitioned to the bottom phase of a polymer/polymer aqueous two-phase system consisting of poly(ethylene glycol) (PEG) and dextran. The partitioning of membranes was monitored by assaying the activity of an inner membrane marker protein and measuring the total protein content in both phases. The enrichment of inner membrane proteins in the dextran phase was also investigated by proteomic methodology, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), trypsin digestion and liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). Using a high level of significance (99.95%) in the subsequent database search, 36 proteins assigned to the inner membrane were identified in the bottom phase, compared to 29 when using the standard sucrose gradient centrifugation method for inner membrane isolation. Furthermore, metal affinity two-phase partitioning was up to 10 times faster than sucrose gradient centrifugation. The separation conditions in these model experiments provide a basis for the selective isolation of E. coli membranes expressing His-tagged proteins and can therefore facilitate research on such membrane proteomes.     

顺-二氯二氨合铂(Ⅱ)与红细胞膜蛋白的相互作用

顺铂可与红细胞膜蛋白结合,并使膜收缩蛋白交联聚合,巯基是顺铂的可能结合部位,顺铂造成膜蛋白构象或在膜内组装的改变,这些结果支持了我们提出的顺铂作用的多靶分子模型。