Dodecylsulfate-induced dissociation of human alpha 2-macroglobulin. An investigation using small-angle neutron scattering and the equilibrium dialysis technique

The dodecylsulfate-induced dissociation of the tetrameric alpha 2-macroglobulin molecule from human plasma has been investigated by the small-angle neutron scattering (SANS) method. The great advantage with the SANS method is that, by using deuterated dodecylsulfate, and contrast variation by changing the D2O/H2O ratio of the solvent, we can selectively study just the protein part, or the dodecylsulfate part, of the protein-dodecylsulfate complex. More than a thousandfold excess of dodecylsulfate (on a molar basis) is needed in order to dissociate alpha 2-macroglobulin to particles with, on average, half the original molecular mass. By combining the SANS data with results obtained by the equilibrium dialysis technique it follows that, under these circumstances, approximately one thousand dodecylsulfate molecules are associated per alpha 2-macroglobulin molecule. From the significant increase in the radius of gyration, which accompanies the dissociation process, we can conclude that the dissociation is associated with a drastic change in conformation of the protein molecule. From measurements where the dodecylsulfate part of the complex dominates the SANS signal we also get an indication that the dodecylsulfate is randomly distributed along the polypeptide chain, rather than being arranged in large clusters at certain regions of the protein molecule. By fitting the parameters of a binding model to the experimental data we obtain the result that most of the more than one thousand bound dodecylsulfate molecules, necessary for dissociation, are involved in the change in conformation, and the dissociation process is, in fact, driven by the binding of a very few extra dodecylsulfate molecules to the dissociation products. These data indicate that the dodecylsulfate-induced dissociation of alpha 2-macroglobulin is probably more complicated than just breaking, for instance, a hydrophobic interaction.     

Small-angle neutron scattering study of the n-decane effect on the bilayer thickness in extruded unilamellar dioleoylphosphatidylcholine liposomes

Dioleoylphosphatidylcholine (DOPC) and n-decane were mixed and hydrated afterwards in an excess of heavy water at 1 wt.% of DOPC. From this dispersion, unilamellar liposomes were prepared by extrusion through polycarbonate filter with 500-A pores. Small-angle neutron scattering (SANS) was conducted on these liposomes. From the Kratky-Porod plot ln[I(Q)Q2] vs. Q2 of SANS intensity I(Q) in the range of scattering vectors Q corresponding to the interval 0.001 A(-2) < or = Q2 < or = 0.006 A(-2), the liposome bilayer radius of gyration Rg and the bilayer thickness parameter d(g) = 12(0.5)Rg were obtained. The values of d(g) indicated that the bilayer thickness is within the experimental error constant up to n-decane/DOPC approximately 0.5 molar ratio, and then increases by 2.4 +/- 1.3 A up to n-decane/DOPC = 1.2 molar ratio.     

Rodlike complexes of a polyelectrolyte (hyaluronan) and a protein (lysozyme) observed by SANS

We study by small-angle neutron scattering (SANS) the structure of hyaluronan -lysozyme complexes. Hyaluronan (HA) is a polysaccharide of 9 nm intrinsic persistence length that bears one negative charge per disaccharide monomer (M(mol) = 401.3 g/mol); two molecular weights, M(w) = 6000 and 500,000 Da were used. The pH was adjusted at 4.7 and 7.4 so that lysozyme has a global charge of +10 and +8, respectively. The lysozyme concentration was varied from 3 to 40 g/L at constant HA concentration (10 g/L). At low protein concentration, samples are monophasic, and SANS experiments reveal only fluctuations of concentration, although, at high protein concentration, clusters are observed by SANS in the dense phase of the diphasic samples. In between, close to the onset of the phase separation, a distinct original scattering is observed. It is characteristic of a rod-like shape, which could characterize "single" complexes involving one or a few polymer chains. For the large molecular weight (500,000), the rodlike rigid domains extend to much larger length scale than the persistence length of the HA chain alone in solution and the range of the SANS investigation. They can be described as a necklace of proteins attached along a backbone of diameter of one or a few HA chains. For the short chains (M(w) ≈ 6000), the rod length of the complexes is close to the chain contour length (～ 15 nm).     

Studies on the microstructure of bile salt aggregates in aqueous n-alkanol solutions by Small Angle Neutron Scattering

Small Angle Neutron Scattering (SANS) measurements on two bile salt micelle systems sodium cholate (NaC) and sodium deoxycholate (NaDC) each 0.1 M in the absence and presence of 0.05 M 1-butanol, 1-pentanol, 1-hexanol and 1-octanol were carried out in D20 at ambient conditions (25 degrees C). The scattering cross-section as a function of wave vector transfer (Q) showed the presence of correlation peaks characteristic of strong inter micellar interactions. The correlation peak positions were shifted to low Q values in the presence of n-alkanols for both NaC and NaDC micelles. Best fit curves of SANS were produced by applying Hayter-Penfold analysis considering monodisperse ellipsoid model for the micelles. The best fits were found for constant semi-minor axis = 8A with an increasing semi-major axis for increasing n-alkanol chain lengths. The micellar growths in presence of n-alkanols were studied using the ESR correlation time measurements on suitable spin probe incorporated micelles. The growth parameter and the hydrodynamic radii values were found to be agreeable with SANS data. Intermicellar interaction potentials seem to increase with the Cn of n-alkanols.     

Isolation and characteristics of small, soluble photoreactive fragments ofRhodospirillum rubrum

A simple isolation procedure for a photochemically active complex from wildtype cells of the photosynthetic non-sulfur bacterium, Rhodospirillum rubrum, is described. The method involves sucrose density centrifugation of chromatophores equilibrated with a large excess of ascorbate and, subsequently, treated with 1% sodium dodecylsulfate in a concentration of 5.4 g/mmole of bacteriochlorophyll. The resulting brown complex has a mol. wt of about 100 000 and a solubility in aqueous buffer of at least 70 mg/ml. 2.7% of the bacteriochlorophyll of the chromatophores was recovered in this preparation. Stoichiometry appears to hold for P870 to cytochrome c₂ (1:1), spirilloxanthin (1:3) and ubiquinone (1:1.7) while cytochrome cc′ was observed in variable amounts. Polyacrylamide gel electrophoresis of this complex using 0.05% sodium dodecylsulfate yielded an even smaller photochemically active fraction (mol. wt approx. 35 000) which contained no cytochrome c₂. The amino acid compositions of both fragments are compared.     

Effect of various denaturing treatments on the structure and activity of a purified CP1 complex

Spinach CP1 complex, purified as previously described [16], was submitted to various dissociating treatments. Chaotropic agents, like urea and thiocyanate salts, remained without effect on the structure and photooxidation of the complex, just SDS at very high concentrations was able to dissociate the chlorophyll from the polypeptides and to abolish the photoreaction. Proteolytic enzymes have no more action on the apparent structure and activity of CP1, but some of them do cleave the large polypeptides (65 kD) into smaller ones, as observed after pigments dissociation. This last result might be an important step in the search for a smaller active P700 protein complex.Abbreviations CP1 pigment-protein complex with the slower mobility in SDS electrophoresis - MW molecular weight - PSI photosystem I - PSII photosystem II - SDS sodium dodecylsulfate     

Small-angle neutron scattering study of the lipid bilayer thickness in unilamellar dioleoylphosphatidylcholine vesicles prepared by the cholate dilution method: n-decane effect

Previous X-ray diffraction studies on fully hydrated fluid lamellar egg phosphatidylcholine phases indicated a approximately 10 A increase of bilayer thickness in the presence of excess n-decane [Biochim. Biophys. Acta 597 (1980) 455], while the small-angle neutron scattering (SANS) on unilamellar extruded dioleoylphosphatidylcholine (DOPC) vesicles detected substantially smaller 2.4+/-1.3 A bilayer thickness increase at n-decane/DOPC molar ratio of 1.2 [Biophys. Chem. 88 (2000) 165]. The purpose of the present study is to investigate the n-decane effect on the bilayer thickness in unilamellar DOPC vesicles prepared by the sodium cholate (NaChol) dilution method. Mixed DOPC+NaChol micelles at DOPC and NaChol concentrations of 0.1 mol/l were prepared in 2H(2)O containing 0.135 mol/l NaCl. This micellar solution was diluted in 0.135 mol/l NaCl in 2H(2)O to reach the final DOPC and NaChol concentrations of 0.008 mol/l. Thirty microliters of n-decane solution in methanol was added to 1 ml of this dispersion. After methanol evaporation, SANS was conducted on the dispersions. From the Kratky-Porod plot ln[I(Q)Q(2)] vs. Q(2) of SANS intensity I(Q) in the range of scattering vector values Q corresponding to interval 0.001 A(-2)相似文献   

Structure of heat-induced beta-lactoglobulin aggregates and their complexes with sodium-dodecyl sulfate

We report on the conformation of heat-induced bovine beta-lactoglobulin (betalg) aggregates prepared at different pH conditions, and their complexes with model anionic surfactants such as sodium dodecyl sulfate (SDS). The investigation was carried out by combining a wide range of techniques such as ultra small angle light scattering, static and dynamic light scattering, small angle neutron scattering, small-angle X-ray scattering, electrophoretic mobility, isothermal titration calorimetry (ITC) and transmission electron microscopy. Three types of aggregates were generated upon heating betalg aqueous dispersions at increasing pH from 2.0 to 5.8 to 7.0: rod-like aggregates, spherical aggregates, and worm-like primary aggregates, respectively. These aggregates were shown not only to differ for their sizes and morphologies, but also for their internal structures and fractal dimensions. The main differences between aggregates are discussed in terms of the ionic charge and conformational changes arising for betalg at different pHs. The formation of complexes between SDS and the various protein aggregates at pH 3.0 was shown to occur by two main mechanisms: at low concentration of SDS, the complex formation occurs essentially by ionic binding between the positive residues of the protein and the negative sulfate heads of the surfactant. At complete neutralization of charges, precipitation of the complexes is observed. Upon further increase in SDS concentration, complex formation of SDS and the protein aggregates occurs primarily by hydrophobic interactions, leading to (i) the formation of an SDS double layer around the protein aggregates, (ii) the inversion of the total ionic charge of each individual protein aggregate, and (iii) the complete redispersion of the protein aggregate-SDS complexes in water. Remarkably, the SDS double layer around the protein aggregates provides an efficient protective shield, preventing precipitation of the aggregates at any possible pH values, including those values corresponding to the isoelectric pH of the aggregates.     

Proton magnetic resonance studies of the binding of an anionic surfactant with a benzene ring to a protein polypeptide with special reference to SDS-polyacrylamide gel electrophoresis.

The binding of an anionic surfactant to a protein polypeptide has been studied by the proton magnetic resonance (PMR) technique to form a part of our studies on the principles of SDS-polyacrylamide gel electrophoresis. Sodium 4-(p-butylphenyl) butane-1-sulfonate (CH3-(CH2)3-0-(CH2)4-SO3-Na+) was employed as an anionic surfactant, and reduced and carbosyamidomethylated (RCAM) bovine serum albumin as a typical protein polypeptide. The binding isotherm of the surfactant to RCAM bovine serum albumin was similar to that of sodium dodecyl sulfate (SDS). The surfactant could replace SDS in SDS-polyacrylamide gel electrophoresis without affecting the wellknown mode of spearation of protein bands. These results gave a sound basis for the assumption that the investigation of the complex between a surfactant with a benzene ring and RCAM bovine serum albumin would provide useful knowledge concerning the principles of SDS-polyacrylamide gel electrophoresis. Aggregation of the aromatic surfactant necessarily brings benzene rings together. A benzene ring is a strong source of the ring current effect on chemical shifts in nuclear magnetic resonance (NMR). Chemical shifts of the surfactant in NMR are, therefore, sensitive to whether the surfactant molecules are single-molecularly dissolved or aggregated. Full advantage was taken of the above fact in the present PMR study of the binding of the surfactant to RCAM bovine serum albumin. The chemical shifts of the phenyl and methyl protons both for the single-molecular and micellar aggregated states were estimated from measurements of the shifts as a function of the surfactant concentration. They shifted to a higher magnetic field on micelle formation, due to the increase of the ring current effect. Corresponding measurements for the complex between the surfactant and RCAM bovine serum albumin gave estimates of the chemical shifts of the phenyl and methyl groups of the surfactant bound to the protein polypeptide. They were found to shift to a magnetic field somewhat higher than that for the micellar state throughout the concentration range of the surfactant examined. These results strongly suggest that the surfactant molecules bind to the protein polypeptide in the form of micelle-like clusters, and that PMR of the groups are further influenced by the diagmagnetic effect of the protein polypeptide present as a core. No appreciable change in the mode of binding, corresponding to the steep increase in the amount of binding in the binding isotherm, was observed from the PMR studies. Taking the observed similarity between SDS and the aromatic surfactant in the binding and the gel electrophoresis into consideration, the present results strongly suggest that SDS also binds to protein polypeptides in the form of micelle-like clusters under the conditions of SDS-polyacrylamide gel electrophoreses, and support our "necklace model".     

Detergent-resistant phospholipase A of Escherichia coli K-12. Purification and properties.

Detergent-resistant phospholipase A, which is tightly bound to the outer membranes of Escherichia coli K-12 cells, was purified approximately 2000-fold to near homogeneity by solubilization with sodium dodecylsulfate and butan-1-ol, acid precipitation, acetone fractionation and column chromatographies on Sephadex G-100 in the presence of sodium dodecylsulfate and on DEAE-cellulose in the presence of Triton X-100. The final preparation showed a single band in the sodium dodecylsulfate gel system. The enzyme hydrolyzes both the 1-acyl and 2-acyl chains of phosphatidylethanolamine or phosphatidylcholine. It also attacks 1-acyl and 2-acylglycerylphosphorylethanolamine. Thus, this enzyme shows not only phospholipase A1 and lysophospholipase L1 activities but also phospholipase A2 and lysophospholipase L2 activities. The enzyme lost its activity completely on incubation at 80 degrees C for 5 min at either pH 6.4 or pH 8.0. It was stable in 0.5% sodium dodecylsulfate at below 40 degrees C. The enzyme was inactivated on incubation for 5 min at 90 degrees C in 1% sodium dodecylsulfate/1% 2-mercaptoethanol/4 M urea. The native and inactivated enzymes showed different protein bands with RF values corresponding to Mr 21 000 and Mr 28 000 respectively, in a sodium dodecylsulfate gel system. Triton X-100 seemed to protect the enzyme from inactivation. The purified enzyme was fully active on phosphatidylethanolamine in the presence of 0.0002% or 0.05% Triton X-100. The enzyme requires Ca2+. From its properties this enzyme seems to be identical with the enzyme purified from crude extracts of Escherichia coli B by Scandella and Kornberg. However, it differs from the latter in its positional specificity and susceptibility to sodium dodecylsulfate. Possible explanation of the difference of positional specificity of the two preparations is also described.     

Multimeric forms of the small multidrug resistance protein EmrE in anionic detergent

Escherichia coli multidrug resistance protein E (EmrE) is a four transmembrane α-helix protein, and a member of the small multidrug resistance protein family that confers resistance to a broad range of quaternary cation compounds (QCC) via proton motive force. The multimeric states of EmrE protein during transport or ligand binding are variable and specific to the conditions of study. To explore EmrE multimerization further, EmrE extracted from E. coli membranes was solubilized in anionic detergent, sodium dodecyl sulphate (SDS), at varying protein concentrations. At low concentrations (≤ 1 μM) in SDS-EmrE is monomeric, but upon increasing EmrE concentration, a variety of multimeric states can be observed by SDS-Tricine polyacrylamide gel electrophoresis (PAGE). Addition of the (QCC), tetraphenyl phosphonium (TPP), to SDS-EmrE samples enhanced EmrE multimer formation using SDS-Tricine PAGE. The relative shapes of EmrE multimers in SDS with or without TPP addition were determined by small angle neutron scattering (SANS) analysis and revealed that EmrE dimers altered in conformation depending on the SDS concentration. SANS analysis also revealed that relative shapes of larger EmrE multimers (≥ 100 nm sizes) altered in the presence of TPP. Circular dichroism spectropolarimetry displayed no differences in secondary structure under the conditions studied. Fluorescence spectroscopy of SDS-EmrE protein demonstrated that aromatic residues, Trp and Tyr, are more susceptible to SDS concentration than TPP addition, but both residues exhibit enhanced quenching at high ligand concentrations. Hence, EmrE forms various multimers in SDS that are influenced by detergent concentration and TPP substrate addition.     

Effect of salt concentration of buffer on the binding of sodium dodecyl sulfate and on the viscosity behavior of the protein polypeptide derived from bovine serum albumin in the presence of the surfactant

The complex between SDS and a protein polypeptide derived from bovine serum albumin was characterized with respect to binding of SDS and viscosity behavior. The amount of bound SDS increased from 1.0 to 2.2 g/g with increase of the buffer concentration from 10 to 220 mM. A logarithmic plot of the amount of bound SDS against the buffer concentration gave a linear relation like in the plot where the number of SDS molecules constituting a spherical micelle of SDS is plotted similarly. The increase in the buffer concentration up to 25 mM, from 25 to 100 mM and beyond 100 mM, was accompanied by a sharp rise, monotonic decrease and levelling-off of the intrinsic viscosity in the respective region. In the region 45-175 mM, a linear relation was found between the intrinsic viscosity and reciprocal square root of the buffer concentration. The observed changes can be interpreted as follows: (1), the electrostatic repulsion between charges introduced by the bound SDS caused the initial increase; (2), shielding of the charges as the result of ion condensation with further increase in ionic strength caused the viscosity drop and subsequent levelling-off. The characteristics of the plots are consistent with the necklace model proposed previously for such complexes in which SDS is bound to a protein polypeptide forming micelle-like clusters and which behave like a flexible polyelectrolyte (Shirahama, K., Tsujii, K. and Takagi, T. (1974) J. Biochem. 75, 309-319).     

Anomalous behavior of bovine alpha s1- and beta-caseins on gel electrophoresis in sodium dodecyl sulfate buffers

Electrophoresis in the presence of sodium dodecyl sulfate (SDS) provides a relatively simple means of determining molecular weights of proteins. This technique relies on the validity of a correlation between some function of Mr and the mobility of the protein through the gel matrix. However, bovine caseins (especially alpha s1-casein) have lower mobilities than expected on the basis of their known Mr. The binding of SDS to both alpha s1-casein (Mr 23,600) and beta-casein (Mr 24,000) reached a maximum at the slightly low value of 1.3 g SDS/g protein. Gel-filtration chromatography showed, however, that the alpha s1-casein:SDS complex was larger than the beta-casein:SDS complex at pH 6.8 or 7.0, but that they were similar in size at pH 2.9 or 3.0. Circular dichroism spectra indicated that the low helical structure content of both alpha s1- and beta-casein increased with the addition of SDS and/or decreasing the pH to 1.5. 13C NMR results showed that SDS bound to alpha s1- and beta-casein in the same way as it did to bovine serum albumin. Either esterification or dephosphorylation followed by amidation of alpha s1-casein increased its mobility in SDS-gel electrophoresis, but neither modification affected beta-casein mobility. These and other results indicate that the low electrophoretic velocity of alpha s1-casein in SDS-gel electrophoresis results from its unexpectedly large hydrodynamic size. This is caused by localized high negative charges on certain segments of alpha s1-casein, which would induce a considerable amount of inter- and intrasegmental electrostatic repulsion, leading to an expanded or extended structure for portions of the alpha s1-casein molecule in the presence of SDS. It is clear that the conformation, and hence the equivalent radius, of an SDS:protein complex is determined by the sequence of amino acids in the protein and that, a priori, it cannot be anticipated that the electrophoretic mobility of such a complex will bear more than a casual relationship to the Mr of the protein.     

Characterization of a new murein-associated lipoprotein in the outer membrane of Proteus mirabilis

A murein-associated outer membrane protein from Proteus mirabilis has been isolated. Since the protein carries ester- as well as amide-linked fatty acids it can be classified as a second outer membrane lipoprotein. An apparent molecular weight of 15,000 for this protein was determined from amino acid analysis and sodium dodecylsulfate/polyacrylamide gel electrophoresis. The amino acid composition, however, does not show similarities with the amino acid composition of the lipoprotein covalently linked to murein, which has a molecular weight of 7,300 as described previously in Proteus mirabilis.Abbreviation SDS sodium dodecylsulfate     

Using small-angle neutron scattering to study the solution conformation of N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates

Our past research developed two N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates that became the first synthetic polymer-anticancer conjugates to be evaluated clinically. The first, FCE28068, contained Dox bound to the polymeric carrier via a tetrapeptidic linker (glycine-phenylalanine-leucine-glycine (GFLG)) (Mw approximately 30,000 g/mol; approximately 8 wt % drug), and the second, FCE28069, contained additionally galactosamine (Gal) (Mw approximately 30,000 g/mol; approximately 7.5 wt % Dox) again bound by a GFLG linker. Galactosamine was included to promote hepatocyte/hepatoma targeting via the asialoglycoprotein receptor. Both conjugates showed antitumor activity and were clinically less toxic than free Dox (2-5 fold). However, despite their similar chemical characteristics, the conjugates displayed a significantly different maximum-tolerated dose (MTD) in patients. The aim of this study, therefore, was to use small-angle neutron scattering (SANS) to explore the solution behavior of a small library of HPMA polymer conjugates including FCE28068, FCE28069, and their pharmaceutical formulations, plus as reference compounds HPMA copolymer-GFLG conjugates containing aminopropanol (Ap) or galactosamine (Gal) alone (i.e., without Dox). The SANS data obtained showed that HPMA copolymer-GFLG-Ap conjugates (containing 5 and 10 mol % side chains) showed evidence of polymer aggregation, however, no indication of aggregation was observed for FCE28068 and FCE28069 over the concentration range studied (2.5-50 mg/mL). Clear differences in the scattering behavior for the two conjugates were observed at equivalent concentration. Data were best fitted by a model for polydisperse Gaussian coils, and the HPMA copolymer-Dox conjugate with Gal (FCE28069) exhibited a larger radius of gyration (Rg) (by approximately 2.5 nm) compared to FCE28068. In conclusion, we have shown that SANS will be a valuable tool to elucidate conformation-performance relationships for polymer-drug conjugates.     

Chlorophyll-protein and polypeptide composition of Mn-deficient sugar beet thylakoids

The chlorophyll-protein and polypeptide composition of manganese deficient and control sugar beet thylakoids was examined using three different detergent-electrophoresis systems. On a per chlorophyll basis, manganese deficiency reduced the amounts of CPa complex (separated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis), and CP 47 and CP 43 complexes (separated by octylglucoside/SDS-polyacrylamide gel electrophoresis) without decreasing the amounts of light harvesting complexes. Lithium dodecylsulfate/Triton X-100 polyacrylamide gel electrophoresis showed that manganese deficiency decreased several thylakoid polypeptides, including a chlorophyll b containing 30 kilodalton chlorophyll-protein complex, but did not decrease the amounts of 28 and 29 kilodalton light-harvesting chlorophyll b-containing polypeptides.     

Solubilization of ribulose-1,5-bisphosphate carboxylase from the membrane fraction of pea leaves

The solubilization of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the membrane fraction was studied in whole leaf extracts and chloroplasts from pea. The amount of membrane-bound Rubisco was dependent on the pH of the chloroplastic lysate buffer. Maximum binding was found at pH 8.0, with about 8% of total leaf Rubisco being bound. The binding of Rubisco to the membranes was strong, and it was not released by repeated washing with hypotonic buffer or by changing ionic strength. Detergents such as Triton X-100, Tween 20, deoxycholate and dodecylsulfate were effective in solubilizing the membrane-bound Rubisco. Triton X-100 was most effective in the range of 0.04% to 0.2% and it solubilized Rubisco from the membrane without any decrease in enzyme activity.Abbreviations BSA bovine serum albumin - CABP carboxyarabinitol-1,5-bisphosphate - DTT dithiothreitol - LDS lithium dodecylsulfate - LHC light-harvesting chlorophyll protein complex - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase - SDS sodium dodecylsulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

High resolution two-dimensional gel electrophoresis of human erythrocyte membrane proteins.

Three different two-dimensional (2-D) gel electrophoretic techniques have been modified to provide high resolution of human erythrocyte membrane proteins. The resulting gels were referenced to the established one-dimensional (1-D) sodium dodecylsulfate (SDS) gel electrophoretic profile, and the effects of endogenous proteolysis and cytosolic contamination were studied. It is concluded that in vitro proteolysis and cytosolic contamination do not contribute significantly to the patterns observed on the 2-D gels, under the conditions used for erythrocyte ghost preparation. The procedures require only small quantities of blood; as many as twenty 2-D gel profiles can be obtained from 5 ml of blood. The combination of nonequilibrium isoelectric focusing (IEF) in the first dimension, SDS electrophoresis in the second dimension, and very sensitive silver staining techniques resolves more than 250 individual protein spots. This appears to be the most useful single procedure for the analysis of red cell membrane proteins. Membrane protein profiles from patients with Duchenne muscular dystrophy, Wernicke-Korsakoff syndrome, and acanthocytosis with degeneration of the basal ganglia were compared with normal controls. The patterns for Duchenne muscular dystrophy and Wernicke-Korsakoff syndrome were not different from normal patterns. The pattern for the patient with acanthocytosis and degeneration of the basal ganglia consistently showed a high level for one protein in the 100,000 mol. wt. range.     

Structural proteins of spring viremia virus of carp

Spring viremia virus (S.V.V.) of carp produced on fathead minnow cells (FHM) has been concentrated by polyethylene glycol and purified by a two step gradient centrifugation. The virus particle has a density of 1.16 in sucrose. Purified S.V.V. was disrupted by sodium dodecylsulfate (SDS), urea, 2-mercaptoethanol and heat, and the proteins were analysed in polyacrylamide gels in presence of SDS. Four different polypeptide chains A, B, C, D were found with M.W. of approximately 150,000, 70,000, 40,000, 19,000 daltons respectively. The three major proteins B, C, D are in molar ratio of 1:4:4.