High-performance liquid chromatographic separation and immunological characterization of soluble bovine viral diarrhea virus antigen

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis of the protein extracts from bovine viral diarrhea virus (BVDV, Singer strain) infected primary calf testicle cells (soluble antigen) showed the presence of four virus specific polypeptides of 105, 90, 84 and 67 kiloDaltons (kD) the 84-kD being the most abundant. Anion-exchange high-performance liquid chromatography (HPLC) of soluble antigen separated the virus specific polypeptides in individual peaks while the gel permeation HPLC collected all of them in a single protein aggregate peak of 290 kD. Except for the 84-kD polypeptide peak in anion-exchange HPLC, all peak fractions were found to be heterogeneous in nature having more than one polypeptide. Analysis of the antisera raised against the peaks having antigen activity showed that antisera against the 84-kD polypeptide peak did not neutralise BVDV while those against the fractions containing the 90- and 105-kD polypeptides neutralised the virus.     

Preparative separation of poliovirus structural polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, copper staining and electroelution, and induction of monospecific antisera

Viral polypeptides were prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by copper staining and electroelution from gel slices. Poliovirus capsid polypeptide VP 1 isolated by this procedure induced monospecific antibodies in rabbits, i.e., antisera reacting only with the homologous polypeptide. Our results demonstrate the applicability of the described copper staining method as a rapid visualization step for preparing viral proteins after SDS-PAGE.     

Analysis of protein/polypeptide interactions in human plasma using nondenaturing micro-2-DE followed by 3-D SDS-PAGE and MS

Previously, we have reported on the analysis of human plasma proteins on a nondenaturing micro-2-DE (mu2-DE) gel, using in-gel digestion followed by MALDI-MS and PMF [1]. Many of the spots on the mu2-DE gel showed apparent masses much larger than the calculated masses of their assigned polypeptides, suggesting noncovalent or covalent interactions between the polypeptides. In the present study, we aimed to further analyze the plasma protein spots on a nondenaturing mu2-DE gel, on which protein/polypeptide interactions have been suggested. The proteins in the spots were extracted under alkaline conditions and subjected to 3-D separation using SDS-PAGE in microslab gel format (muSDS gel) with or without the sample treatment of reduction-alkylation. The clear bands in each lane of the muSDS gels demonstrated the successful extraction of proteins from the relevant gel spot and visualized the relative contents of the polypeptides in the spot. Most of the bands were assigned by in-gel digestion followed by MALDI-MS and PMF (MASCOT/Swiss-Prot). The large discrepancy between the apparent mass value of a protein spot and the estimated mass values of the polypeptide bands on a nonreducing muSDS gel strongly suggested noncovalent polypeptide interactions. The differences in the polypeptide separation patterns on the muSDS gels, between with and without the treatment of reduction-alkylation, confirmed polypeptide disulfide bonding. The method employed here, aiming to integrate information on the proteins separated on nondenaturing 2-DE gels with that on the interactions between polypeptides, would help the comprehensive understanding of complex protein systems.     

Noncovalent interactions in human plasma proteins analyzed by the comparison of nondenaturing and denaturing micro-2-D gel electrophoresis patterns after polypeptide assignment using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass fingerprinting

Previously, we reported the analysis of human plasma proteins by 2-DE under nondenaturing conditions (Type-I 2-DE) followed by the assignment of stained spots using MALDI-MS and PMF [1]. Here, we employ 2-DE conditions modified only in the second-dimensional separation; SDS was added in the gradient slab gel aiming to dissociate noncovalently bound proteins/polypeptides (Type-II 2-DE). Totally 169 CBB-stained spots on a micro-2-DE gel were numbered and subjected to polypeptide assignment using MALDI-MS-PMF. One hundred sixty spots out of the 169 provided significant match (p <0.05) with polypeptides in databases. Comparisons of the results of polypeptide assignment on the two 2-DE patterns indicated that 10 polypeptides in 20 stained spots on the Type-I 2-DE pattern [1] shifted toward low-molecular-weight positions on the Type-II 2-DE pattern, demonstrating the presence of noncovalent interactions. Seventeen polypeptides in 38 stained spots were only assigned on the Type-II 2-DE gel, which could mostly be accounted for by the disruption of noncovalent protein-protein interactions in the presence of SDS, i.e., protein/polypeptide complexes which might form smear bands on the Type-I 2-DE gel dissociate to form clear spots on the Type-II 2-DE gel. The method employed here, comparisons of nondenaturing and denaturing 2-DE maps with polypeptide assignment by MALDI-MS-PMF, would enable the simultaneous detection of multiple noncovalent interactions in complex protein/polypeptide systems.     

Two-dimensional electrophoresis of the total polypeptides in ripe red grape berries.

The total polypeptide composition of mature grape berries was analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis (isoelectric focusing in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension), followed by Coomassie Blue and nitrate silver staining, respectively. Adapted methods for total protein preparation of grapes and for two-dimensional gel electrophoretic separation of polypeptides are presented. The grape patterns presented up to 52 fractions with Mrs ranging from 15,000 to 110,000. The polypeptides displayed pIs from 4.6 to 7.3. A group of spots from Mr 28,000 to 83,000 and with a pI from 4.6 to 5.4 was strongly silver stained. The Mr 28,000 spot, pI 4.6, was revealed to be a complex of four fractions. Reproducible separations were obtained with the different carrier ampholyte mixtures tested.     

Analysis of cerebrospinal fluid from patients with psychiatric and neurological disorders by two-dimensional electrophoresis: identification of disease-associated polypeptides as fibrin fragments

Two-dimensional electrophoresis (2-DE) of cerebrospinal fluid (CSF) samples--from 347 patients with various psychiatric and neurological disorders--and subsequent silver staining revealed two additional polypeptides (Mr 40,000) in 49% of 111 schizophrenics, 46% of 43 schizoaffective patients, 36% of 41 patients with affective disorders, 43% of 28 patients with multiple sclerosis, but not in 25 patients without neurological symptomatology, nor in 9 patients with Lues, and in only 2 of 25 patients with AIDS. The two polypeptides, as detected by 2-DE, eluted after size exclusion chromatography in fractions containing proteins with Mr greater than 200,000. After 2-DE of CSF samples, enriched by gel chromatography, the polypeptides were immobilized by blotting onto glass-fiber membranes and subjected to N-terminal sequencing. Polypeptide A was identified as beta-chain remnant (beta 2), derived from plasmin cleavage of fibrin(ogen). After size exclusion chromatography, 2-DE, and Western blotting, polypeptide A and B, as well as several other spots, reacted with fibrinogen antibodies, suggesting that the polypeptides are subunits of a fibrin degradation complex.     

COMPARISON OF PHOTOTRAP COMPLEXES FROM CHROMATOPHORES OF RHODOSPIRILLUM RUBRUM, RHODOPSEUDOMONAS SPHEROIDES, AND THE R-26 MUTANT OF RHODOPSEUDOMONAS SPHEROIDES*

Abstract— After dissolution of the membrane structure of chromatophores from Rhodospirillum rubrum, Rhodopseudomonas spheroides , and the R-26 mutant of Rhodopseudomonas spheroides , active phototrap complexes from each have been purified by a column electrophoresis procedure. Phospholipids and transition metals were well separated from the phototrap complex in all three systems. The purified R. rubrum phototrap complex retained a full complement of antenna bacteriochlorophyll and carotenoid pigments which had nearly the same absorbance spectra as in the intact cell, and which delivered absorbed light energy to the phototrap with just as high efficiency as in the intact cell. Sodium dodecyl sulfate (SDS) disc gel electrophoresis using Tris buffer showed that these preparations often contained only two prominent polypeptides of 30,000 ± 2000 and 12,000 ± 4000 mol. wt., and a lesser amount of a third polypeptide of 21,000 ± 2000 mol. wt.
The phototrap complexes prepared from the wild type and the R-26 mutant of R. spheroides were similar, in that a partial separation from antenna pigments occurred during column electrophoresis. Both complexes had prominent polypeptides of 24,000 ± 2000 and 21,000 ± 2000 mol. wt., but no polypeptide of 30,000 mol. wt remained after electrophoresis. A third major polypeptide occurred with a mol. wt. of about 12,000 but seemed identifiable with an incompletely separated antenna pigment fraction. The phototrap complex prepared from the R-26 mutant had a typical reaction center spectrum.
In the case of wild type R. spheroides purification, two distinct protein-pigment complexes separated. Although the absorbance of the bacteriochlorophyll and carotenoid pigments were little changed from those of the in vivo system, different polypeptides in the two fractions were observed by SDS disc gel electrophoresis; only one fraction seemed to be intimately related with the phototrap complex.     

Experimental Studies with a Bonded N-acetylaminopropylsilica Stationary Phase for the Aqueous High Performance Exclusion Chromatogrpahy of Polypeptides and Proteins

Abstract

The potential of the micoparticulate, chemically bonded N-acetylaminopropylsilica stationary phase of nominal pore diameter of 100 angstroms in the high speed gel permeation chromatography of polypeptides and small proteins has been further investigated. The influence of ionic strength on the elution behaviour of a selected group of polypeptides and proteins on this bonded hydrophilic support has been examined. The results obtained with this porous, microparticulate bonded ‘amide’ phase silica support, packed into standard analytical-size stainless steel HPLC columns, indicate that milligram quantities of polypeptides and protein swith molecular mass up to 45,000-50,000 daltons can be efficiently fractionated with excellent recoveries of biological activities. The role of silica-based sorbents in the gel permeation fractionation of polypeptide and protein hormones, including those of pituitary and hypothalamic origin, is discussed.     

Assignment of human plasma polypeptides on a nondenaturing 2-D gel using MALDI-MS and PMF and comparisons with the results of intact protein mapping

Human plasma proteins were separated by 2-DE under nondenaturing conditions followed by the assignment of the CBB-stained spots using MALDI-MS and PMF, aiming to correlate the information of intact proteins with that of constituent polypeptides. A microgel system was employed to facilitate the analysis. Totally 157 spots on a nondenaturing micro-2-DE gel were numbered, the spots were excised, the proteins in the gel pieces were subjected to in-gel digestion with trypsin followed by polypeptide analysis using MALDI-MS and PMF. Two PMF algorithms, MASCOT (with Swiss-Prot database) and ProFound (with NCBInr database) were employed. A total of 153 spots out of the 157 provided significant match (p <0.05) with polypeptides in databases. Eighty spots were assigned to contain multiple (2-4) polypeptides, suggesting (i) noncovalent interaction between proteins/polypeptides, (ii) disulfide bonding of polypeptides, or (iii) overlapping of the protein locations on the gel. The results of polypeptide assignment coincided very well with the results of protein mapping previously reported, in which 33 plasma proteins were identified using blotting-immunochemical staining (Manabe, T., Takahashi, Y., Higuchi, N., Okuyama, T., Electrophoresis 1985, 6, 462-467). Further, 19 polypeptides in 25 spots were newly assigned. These results demonstrate that the techniques of MALDI-MS and PMF can be applied for analysis of proteins separated on nondenaturing 2-DE gels, providing information on their polypeptide structure. The integrated information on proteins and polypeptides would help the comprehensive understanding on the functions of complex protein systems.     

Multiple polypeptide forms observed in two-dimensional gels of Methylococcus capsulatus (Bath) polypeptides are generated during the separation procedure

We have examined two-dimensional electrophoresis (2-DE) gel maps of polypeptides from the Gram-negative bacterium Methylococcus capsulatus (Bath) and found the same widespread trains of spots as often reported in 2-DE gels of polypeptides of other Gram-negative bacteria. Some of the trains of polypeptides, both from the outer membrane and soluble protein fraction, were shown to be generated during the separation procedure of 2-DE, and not by covalent post-translational modifications. The trains were found to be regenerated when rerunning individual polypeptide spots. The polypeptides analysed giving this type of trains were all found to be classified as stable polypeptides according to the instability index of Guruprasad et al. (Protein Eng. 1990, 4, 155-161). The phenomenon most likely reflects conformational equilibria of polypeptides arising from the experimental conditions used, and is a clear drawback of the standard 2-DE procedure, making the gel picture unnecessarily complex to analyse.     

Specific Proteins in the Indirect Somatic Embryogenesis of Freesia Refracta

IntroductionHaberlandt[1]proposed the conception of plantso-matic embryo in 1902, which states that plant cellsexhibit totipotency; each cell could divide ceaselesslyand eventually develops into a mature plantlet. Allthese provide a theoretical evidence for somatic embryo-genesis. The related researches reveal thatplantembry-ogenesis occurs as a result of the selective expression ofsets of genes that allowthe synthesis of special proteinsin a temporal and spatial sequential manner under themut…     

THE RESOLUTION OF CHLOROPHYLL a/b BINDING PROTEINS BY A PREPARATIVE METHOD BASED ON FLAT BED ISOELECTRIC FOCUSING

We describe a new fractionation method for intrinsic membrane proteins based on flat bed isoelectric focusing (IEF) in granulated gel. The characteristics of the separation in the presence of the non-ionic detergent dodecylmaltoside are considered. The method has been applied to the fractionation of chlorophyll a/b binding proteins from chloroplast grana membranes. Several Light Harvesting Complexes II (LHC II) have been resolved showing differences in their polypeptide composition. Probing with monoclonal and polyclonal antibodies showed that polypeptides belonging to different [EF fractions with the same mobility in denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis, are immunologically distinct polypeptides. This is the first report of the presence in the thylakoid membrane of a number of LHCII polypeptides that may reflect the genetic complexity of the Cab genes. Moreover preparative amounts have been obtained of the minor chlorophyll a/b proteins CP 29, CP 26 and CP 24 that have been recently described. The analysis of a currently used LHCII preparation by the present method shows that this fraction is actually contaminated by two minor chlorophyll a/b proteins.     

Preparative isoelectric focusing of reduced wheat gluten proteins

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.     

Stimuli‐Triggered Sol–Gel Transitions of Polypeptides Derived from α‐Amino Acid N‐Carboxyanhydride (NCA) Polymerizations

The past decade has witnessed significantly increased interest in the development of smart polypeptide‐based organo‐ and hydrogel systems with stimuli responsiveness, especially those that exhibit sol–gel phase‐transition properties, with an anticipation of their utility in the construction of adaptive materials, sensor designs, and controlled release systems, among other applications. Such developments have been facilitated by dramatic progress in controlled polymerizations of α‐amino acid N‐carboxyanhydrides (NCAs), together with advanced orthogonal functionalization techniques, which have enabled economical and practical syntheses of well‐defined polypeptides and peptide hybrid polymeric materials. One‐dimensional stacking of polypeptides or peptide aggregations in the forms of certain ordered conformations, such as α helices and β sheets, in combination with further physical or chemical cross‐linking, result in the construction of three‐dimensional matrices of polypeptide gel systems. The macroscopic sol–gel transitions, resulting from the construction or deconstruction of gel networks and the conformational changes between secondary structures, can be triggered by external stimuli, including environmental factors, electromagnetic fields, and (bio)chemical species. Herein, the most recent advances in polypeptide gel systems are described, covering synthetic strategies, gelation mechanisms, and stimuli‐triggered sol–gel transitions, with the aim of demonstrating the relationships between chemical compositions, supramolecular structures, and responsive properties of polypeptide‐based organo‐ and hydrogels.     

An improved sodium dodecyl sulfate-polyacrylamide gel electrophoresis system for the analysis of membrane protein complexes

Membrane protein complexes such as the reaction center complexes of oxygenic photosynthesis or the complex I of mitochondira are composed of many subunit polypeptides. To analyze their polypeptide compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a wide range of molecular sizes has to be resolved, especially in the low molecular mass range. We have improved the traditional Tris/HCI buffer systems adopting a Tris/2-(N-morpholino)ethanesulfonic acid (MES) buffer system containing 6 M urea. This gel system was used with an 18-24% acrylamide gradient for the separation of polypeptides with molecular masses from below 5 kDa to over 100 kDa. This buffer system can also be applied to the usual uniform concentration of acrylamide gel and also to minislab gels.     

Separation of the Secretin-Glucagon Family Peptides by RP-HPLC in Triethylammonium Phosphate Buffers

Abstract

Four preparations of hormonal polypeptides related to the secretin-glucagon family (secretin, glucagon, vasoactive intestinal polypeptide, gastric inhibitory polypeptide) and a preparation of pancreatic polypeptide are analyzed by reverse-phase HPLC on a μ-Bondapack column in triethylammonium phosphate buffers. The chromatographic results reveal the presence of impurities in natural peptides (VIP, GIP) and in synthetic secretin. Moreover, an important relative difference in apparent hydrophobic interactions is showed between VIP and the others peptides investigated. When a mixture of these five polypeptides is chromatographed in a 7 minute concave gradient buffer of 26% to 31% acetonitrile, VIP, GIP, S, G and PP are successively eluted in a total time of less than 20 minutes. This elution sequence does not fit with predicted retention times calculated according to the method proposed by Meek (1).     

梅花鹿茸多肽的化学结构及生物活性

在马鹿茸活性多肽结构与功能研究基础上, 从新鲜梅花鹿茸中分离纯化了活性单体多肽, 确定了其化学结构, 并与马鹿茸多肽进行结构与活性比较. 利用离子交换层析、 凝胶过滤层析及反相高效液相色谱层析等生物化学技术, 从梅花鹿茸中分离得到1个新多肽, SDS-PAGE电泳显示为一条带, HPLC图谱为单一峰, MALDI-TOF MS给出该多肽的精确分子量为3263.4, 其等电点pI=8.15. 一级结构研究表明, 该多肽是由32个氨基酸残基组成的直链多肽, 不含半胱氨酸, 富含缬氨酸、 赖氨酸、 亮氨酸和甘氨酸, 氨基酸序列为VLSATDKTNVLAAWGKVGGNAPAFGAEALERM. 生物活性检测结果表明, 该多肽可促进原代培养的表皮细胞和软骨细胞增殖, 也能刺激NIH3T3成纤维细胞株的分裂. 梅花鹿茸多肽与马鹿茸多肽在结构上均为32个氨基酸残基组成的直链多肽, 但第5, 8, 11和30位氨基酸残基不同. 2种多肽结构上的变化并未影响其促细胞增殖生物活性.     

Investigation of the protein component of the lipopolysaccharide-protein complex ofYersinia pseudotuberculosis

It has been established by electrophoresis in polyacrylamide gel that the protein component of the lipopolysaccharide-protein complex ofYersinia pseudotuberculosis consists of two poly-peptides with molecular weights of 42,000–45,000 and 18,000–20,000. These polypeptides have been detected in the cell wall ofY. pseudotuberculosis as the main components. Protein fractions B-2 and B-3, differing with respect to solubility in phenol, were isolated from the LPPC ofY. pseudotuberculosis by extraction with hot 45% phenol. These protein fractions had the same polypeptide composition, which is characteristic for membrane proteins. B-3 included the LPS component and B-2 contained lipid A or fragments of it as impurity. Fractions B-3 and B-2 precipitated with antiserum to the LPPC.Pacific Ocean Institute of Bioorganic Chemistry of the Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 92–97, January–February, 1980.     

Occurrence of Polypeptides in Other Organisms Cross-reacting with Antibodies Against A. Xylinum Cellulose Synthase

Antibodies (anti-83 and anti-93) against the cellulose synthase complex from A. xylinum ATCC 53582 have been employed to study the evolutionary conservation of this enzyme complex among various A. xylinum strains, selected species of other cellulose- producing bacteria, algae, and vascular plants. Of the 18 A. xylinum strains examined, the 83 Kd polypeptide clearly is detected only in 4 strains while the 93 Kd polypeptide is observed in all 18 strains. Assuming that the revised acsAB gene (Saxena et al., 1994) encoding the 83 and 93 Kd polypeptides as a single polypeptide holds true for all A. xylinum strains, it is proposed that the cellulose synthase is conserved in A. xylinum but with varying degrees of homology. An unknown regulatory mechanism causing the degradation of the 83 Kd polypeptide in response to agitated culturing conditions has been suggested to explain the absence of the 83 Kd polypeptide in most of the Acetobacter strains examined. A. xylinum cellulose synthase appears to be conserved in phylogenetically related Rhizobium and Agrobacterium species, but not in algae and plants.     

液相色谱法分离胃蛋白酶原

首次应用高压凝胶过滤色谱和中压阴离子交换色谱提纯胃蛋白酶原,得到抗原ＰＧⅠ和ＰＧⅡ,一次提纯仅需５０ｍｉｎ。与以往文献报道的胃蛋白酶原纯化方法相比,具有简易、快速、高效的特点。