Assessment study on the high-performance liquid chromatography-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate.

The HPLC-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate (SDS) was assessed with special attention to the behavior of the surfactant. A significant amount of SDS was found to be adsorbed to the hydroxyapatite packed in the column from the starting buffer, 50 mM sodium phosphate buffer, pH 7.0, only when the buffer contained SDS in a concentration at or above its critical micelle concentration. When the phosphate buffer concentration was increased while the SDS concentration was kept at 1 mg/ml, the adsorbed surfactant was desorbed in advance of the release of proteins. Polypeptides derived from proteins could be successfully separated only when the column had been thoroughly equilibrated with the above-mentioned starting buffer solution. When a protein polypeptide complexed with SDS, which had been similarly equilibrated, was applied to the column, an amount of SDS corresponding to 75-90% (w/w) of the surfactant originally bound to the polypeptide was released upon its binding to the hydroxyapatite. On the other hand, porin, an Escherichia coli outer membrane protein, retaining its trimeric native structure in the presence of SDS, released a significantly smaller amount of SDS. When the membrane protein was denatured to give a single polypeptide, it behaved in a manner similar to that of the other protein polypeptides. The mechanism of binding of the protein polypeptides was discussed on the basis of these results. The native and denatured entities of porin could be efficiently separated as the result of the difference in their mode of interaction with the hydroxyapatite.     

Studies on algal cytochromes VI: some properties and amino acid sequence of cytochrome c6 from a green alga, Bryopsis maxima

A photosynthetic c-type cytochrome, cytochrome c6, was extracted from a green alga, Bryopsis maxima, by cutting and immersing the frozen thalli in phosphate buffer, pH 7.0, and purified by acrinol treatment, ammonium sulfate fractionation, DEAE-Sephacel chromatography and Bio-Gel P-10 gel filtration. The ferrcytochrome c6 has absorption maxima at 553.5 (alpha), 523 (beta), 417 (gamma), 318 (delta), and 275 nm, and the ferricytochrome at 695, 528, and 411 (gamma). The molecular weight was estimated to be about 10,000 from Sephadex G-75 gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The midpoint redox potential for the cytochrome was determined by equilibrium titration with a ferro- and ferricyanide system to be 0.385 volt at pH 7.0. Isoelectric points for ferro- and ferricytochromes were determined by density gradient isoelectric focusing electrophoresis to be at pH 3.91 and 4.02, respectively. The complete amino acid sequence of the cytochrome was determined by Edman degradation and by carboxypeptidase digestions of the Cm-cytochrome, 6 staphylococcal protease peptides and 5 lysyl endopeptidase peptides. The cytochrome contained 88 amino acid residues, giving a molecular weight of 9,904 including 1 mol of heme c. The sequence is as follows: GGDLEIGADVFTGNCAACHAGGANSVEPLKTLNKEDVTKYLDGGLSIEAITSQVRNGKGAMPAWSDRLD DEEIDGVVAYVFKNINEGW. A phylogenetic tree of 13 algal cytochromes c6 was constructed by comparing the amino acid differences.     

High-performance liquid chromatography of apolipoproteins in serum high-density lipoproteins

A simple and rapid method for apolipoprotein analysis in serum high-density lipoproteins (HDL) has been developed using high-performance liquid chromatography (HPLC) with sodium phosphate buffer (pH 7.0) containing 0.1% sodium dodecyl sulphate (SDS) as eluent. In contrast to the use of urea solution as an eluent, apolipoproteins can be analysed by applying an incubation mixure of HDL and the eluent buffer. A TSK-GEL column of G3000SW was found to be more profitable than G2000SW or G4000SW for analysis of HDL apolipoproteins. Elution patterns monitored by absorbance at 280 nm using a G3000SW column can give precise quantitative as well as qualitative information about apolipoproteins of molecular weight between 10⁴ and 10⁵. HPLC patterns of HDL apolipoproteins were compared between individual human subjects with various diseases. Elution profiles for lipid components in an incubation mixture were also examined.     

Effect of various detergents on protein migration in the second dimension of two-dimensional gels.

Two-dimensional gel electrophoresis (2D)1 is a powerful technique used to separate complex protein mixtures. The technique involves the separation of proteins by charge in the first dimension and by molecular weight in the second dimension. The effect of substituting various detergents for sodium dodecyl sulfate (SDS) in the second dimension (PAGE) was investigated. Individual C-10 through C-14 alkyl sulfates, C-11 through C-14 alkyl sulfonates, sodium N-lauroyl-N-methyl-taurine, N-lauroylsarcosine, sodium laurate, or benzyldimethyl-n-hexadecylammonium chloride were substituted for SDS in equilibration buffer, gel buffer, and upper running buffer. The cationic benzyldimethyl-n-hexadecylammonium chloride system was run with reversed polarity. Dramatic effects on protein migration from human mesothelial cell extracts were observed when different detergents were utilized. The C-12 (SDS) through C-14 alkyl sulfates and sulfonates resulted in anomalous migration of the simple epithelial keratins. Unlike SDS, the C-10 and C-11 alkyl sulfates and C-11 sulfonate resulted in gels in which the keratins were separated accurately with respect to their gene sequence-determined molecular weights. However, with these shorter chain alkyl sulfates and sulfonate, resolution was compromised, especially with respect to the high-molecular-weight polypeptides. The C-12 alkyl sulfate (SDS) and alkyl sulfonate provided the best resolution of polypeptides. Mixtures of C-11 sulfate and SDS resulted in gels with better sequence molecular weight estimates and high resolution. In addition, trace amounts of sodium tetradecyl sulfate/sodium heptadecyl sulfate in commercial SDS preparations had an effect on polypeptide resolution.     

Degradative acetolactate synthase of Bacillus subtilis: purification and properties.

A degradative acetolactate synthase (acetolactate pyruvate-lyase [carboxylating], EC 4.1.3.18) from Bacillus subtilis has been partially purified and characterized. The synthesis of the enzyme was induced by growth of cells in minimal medium plus isobutyrate or acetate. The enzyme was partially purified by ammonium sulfate fractionation, gel filtration, and hydroxyapatite chromatography. The pH optimum of the purified enzyme was 7.0 in phosphate buffer. When assayed in phosphate buffer (pH 7.0), activity was stimulated by acetate and inhibited by sulfate. When assayed in acetate buffer (pH 5.8), activity was inhibited both by sulfate and phosphate. Michaelis-Menten kinetics was observed when the enzyme was assayed in phosphate buffer (pH 6.0 or 7.0), and inhibition by sulfate was competitive and activation by acetate was noncompetitive. When assayed in acetate buffer (pH 5.8), nonlinear Lineweaver-Burk plots were obtained; inhibition by phosphate appeared to be competitive and that by sulfate was of the mixed type. The approximate molecular weight of the purified enzyme was 250,000 as determined by gel filtration.     

Isolation of alpha-connectin, an elastic protein, from rabbit skeletal muscle

alpha-Connectin (also called titin 1) has been isolated from rabbit back muscle. Myofibrils were well washed with 5 mM NaHCO3 and then extracted with 0.2 M sodium phosphate, pH 7.0. The extract was dialyzed against 0.1 M potassium phosphate, pH 7.0, to sediment myosin. The supernatant, adjusted to 0.18 M potassium phosphate, pH 7.0, and 4 M urea, was subjected to DEAE Toyopearl column chromatography. beta-Connectin was eluted in the flow-through fraction and alpha-connectin was eluted at around 0.1 M NaCl, when a 0 to 0.25 M NaCl gradient was applied. The separated alpha-connectin was dialyzed against 0.2 M potassium phosphate, pH 7.0. The resultant alpha-connectin showed the same mobility as that in an SDS extract of rabbit back muscle on SDS gel electrophoresis using 1.8% polyacrylamide gels. A monoclonal antibody against chicken breast muscle beta-connectin reacted with the alpha-connectin isolated from rabbit back muscle.     

Purification and properties of pig brain guanine deaminase.

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) from pig brain was purified to homogeneity by column chromatography and ammonium sulphate fractionation. Homogeneity was established by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS). The molecular weight of 110 000 was determined by gel filtration and sucrose density gradient centrifugation. SDS polyacrylamide gel electrophoresis indicated subunits of a molecular weight of 50 000. The amino acid composition, the isoelectric point and the number of -SH groups were determined. 5.5'-Dithiobis-(2-nitrobenzoic acid) reacts with about seven -SH groups in the native enzyme, but upon denaturation with SDS, 10 -SH groups react with this former reagent. Using electrolytic reduction, 44 half-cystines were determined in accordance with the number of cysteic acid residues determined by amino acid analysis after performic acid oxidation. The Km values determined for substrates of the enzyme were 1.1 . 10(-5) M for guanine in 0.1 M Tris. HCl buffer (pH 8.0) and 3.3 . 10(-4) M for 8-azaguanine in 0.1 M phosphate buffer, pH 6.4. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 6.2 and pH 8.2. The chemical and kinetic evidence suggests that cysteine and histidine may be essential for the catalysis.     

Influence of salts on the activity and the subunit structure of ornithine decarboxylase from rat liver

The influence of salts on the subunit structure and the kinetics of purified rat ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was examined. Salts were found to cause subunit dissociation of the enzyme, producing the monomeric form of molecular weight 55 000 in the presence of 0.25 M NaCl/10 mM sodium phosphate buffer (pH 7.0): the molecular weight was estimated to be 150 000 in 10 mM and 250 000 in 1 mM sodium phosphate buffer. Inclusion of NaCl in kinetic assays of rat ornithine decarboxylase had little effect on maximal velocity. However, the Km value for L-ornithine was dramatically increased with increasing sodium chloride concentration: the presence of 0.25 M NaCl resulted in a 10-fold increase of the Km. Thus, the presence of salts caused dramatic changes both in the subunit structure and in the catalytic property of the enzyme, although a direct correlation between both the changes was not evidenced.     

Studies on interaction of oligopeptides with sodium dodecyl sulfate: Stopped-flow kinetics of chemical modification of tryptophan residues withN-bromosuccinimide

The stopped-flow kinetics of the reaction between oligopeptides containing tryptophan residues andN-bromosuccinimide (NBS) were studied in 50 mM sodium phosphate buffer (pH 7.0) containing sodium dodecyl sulfate (SDS). Decreases in the reaction rates attributable to the interaction between oligopeptides and SDS were observed, and oligopeptides studied were classified into types I and II on the basis of the interaction modes. Type I oligopeptides were dissolved in SDS micelles; type II oligopeptides interacted cooperatively with SDS monomers. The manner of interaction between SDS and oligopeptides of type II could be interpreted by a simple equilibrium relation: oligopeptide+n·(SDS)=oligopeptide·(SDS) _n .     

Studies on interaction of oligopeptides with sodium dodecyl sulfate: Stopped-flow kinetics of chemical modification of tryptophan residues withN-bromosuccinimide

The stopped-flow kinetics of the reaction between oligopeptides containing tryptophan residues andN-bromosuccinimide (NBS) were studied in 50 mM sodium phosphate buffer (pH 7.0) containing sodium dodecyl sulfate (SDS). Decreases in the reaction rates attributable to the interaction between oligopeptides and SDS were observed, and oligopeptides studied were classified into types I and II on the basis of the interaction modes. Type I oligopeptides were dissolved in SDS micelles; type II oligopeptides interacted cooperatively with SDS monomers. The manner of interaction between SDS and oligopeptides of type II could be interpreted by a simple equilibrium relation: oligopeptide+n·(SDS)=oligopeptide·(SDS) _n .     

Characterization of polypeptide chains of epidermal prekeratin and reconstituted filaments

Prekeratin was isolated from bovine snout epidermis with 0.1 M citric acid/sodium citrate buffer, pH 2.6 (buffer A). Filaments, 6.0-9.0 nm wide, were produced by dialysis against low ionic strength buffer A or by dissociating prekeratin in 8 M urea solution followed by dialysis against 0.005 M Tris-HCl buffer, pH 8.0. The polypeptide composition of both prekeratin and filaments was studied by four different SDS-polyacrylamide gel electrophoresis methods. The best resolution was obtained by Laemmli's technique in which both prekeratin and filaments were separated into three major and seven distinct minor bands of polypeptides. The major ones comprise approx. 70% of total polypeptides and their estimated molecular weights are 68 000, 54 000, and 50 000. The molecular weight of minor ones is in decreasing order 65 000, 63 000, 61 000, 58 000, 47 000, 44 000 and 42 000. It is proposed that the major polypeptides form the backbone structure of epidermal filaments and the minor polypeptides play a role in its stabilization.     

Reconstitution of rapeseed high molecular weight protein from isolated subunits

The high molecular weight protein was isolated from rapeseed and characterised. Six subunits were isolated in SDS (0.01%) solution on polyacrylamide-gel electrophoresis and by gel filtration on Sephadex G-100. Reassociation by removing SDS by co-dialysis, against 10 mM sodium phosphate buffer (pH 7.9) was done and the yield was about 90%. The reconstituted protein was indistinguishable from the intact protein in all respects. The subunits isolated from the native protein and the reconstituted protein were found to have identical molecular weights and N-terminal amino acids. No disulphide bonds were observed in the subunit association. Amino acid analysis of the proteins and the six subunits was performed and the number of each amino acid residue calculated.     

The effect of sodium dodecyl sulfate on the structure and function of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) has been shown to exist as a dimer of molecular weight of 23,000 in 25 mm phosphate buffer, at pH 7.0 (the ionic strength 0.1 m with NaCl), 25.0 °C in the concentration range of 0.01–10 mg/ml. In the present paper, the effects of an anionic detergent, sodium dodecyl sulfate (SDS), on the structure and function of SSI has been examined, [a]The molecular weight of SSI was measured in the SDS solution with the sedimentation equilibrium method of the multicomponent-polydisperse system under the conditions described above, and thereby it has been shown that SSI dissociates into monomers with SDS of 0.03–0.12% ($\text{w}\text{v}$) when the concentration of SSI is 1.00 mg/ml (87.0 μm as monomer), [b]As SSI dissociates into monomers, there were observed blue-shift troughs at 293 nm and 300 nm due to a tryptophyl residue and a red-shift of phenylalanyl residues in the absorption difference spectrum induced by the binding of SSI and SDS. [c] The inhibitory activity of SSI against subtilisin BPN′-catalyzed hydrolysis of p-nitrophenyl acetate was measured under the conditions that SSI is in monomer in the SDS solution. Unexpectedly half of the inhibitory activity of SSI against subtilisin BPN′ is lost in the SDS solution.     

Chiral separation of vinpocetine using cyclodextrin-modified micellar electrokinetic chromatography

A cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) technique has been developed for enantioseparation of vinpocetine using an inexpensive 2-hydroxypropyl-β-CD (HP-β-CD) as the chiral selector (CS). The best chiral separation was achieved using 40 mM HP-β-CD as the CS in 50 mM phosphate buffer (pH 7.0) consisting of 40 mM sodium dodecyl sulfate (SDS) at a separation temperature and separation voltage of 25°C and 25 kV, respectively. To the author's best knowledge, this is the first CD-MEKC study able to successfully separate the four stereoisomer of vinpocetine in separation time of 9.5 min and resolution of 1.04-3.87.     

Purification and characterization of bifunctional alginate lyase from Alteromonas sp. strain no. 272 and its action on saturated oligomeric substrates

A marine bacterium (strain No. 272) isolated from sea mud in Omura Bay produced an alginate lyase and was classified as an Alteromonas species. The enzyme was purified from the culture medium of the bacterium by DEAE-Cellulofine, Sephadex G-100 gel chromatography to an electrophoretically homogeneous state in the presence and absence of SDS. The molecular mass of the enzyme was 23 and 33.9 kDa on Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis, respectively, with an isoelectric point of 3.8. The predominant secondary structure of the enzyme was found to be most likely beta-structure by circular dichroism. The enzyme was most active at pH 7.5-8.0 and stable around pH 5-11. The enzyme was more labile in Tris-HCI buffer (pH 7.0) to heat treatment, than in phosphate buffer (pH 7.0). No of metal ions significantly affected the enzyme activity. The enzyme acted on sodium alginate in an endo-type manner and on two components of alginate, poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate, as judged by routine ultraviolet assay (235 nm) and circular dichroic spectral changes of the substrates. However, the coexisting poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate apparently interacted with the enzyme in a competitive manner. Although the enzyme depolymerized alginate in an endo-type, it did not act on trimeric guluronate and mannuronate, but on the tetramers or more. The kinetic analyses showed that kcat/Km for each oligomer was larger for the guluronate oligomers than for the mannuronate ones, and that the subsite structure of the enzyme most likely consisted of six binding sites from the intrinsic reaction rate constant (kint) and intrinsic substrate binding constant (Kint).     

Purification and properties of L-alpha-glycerophosphate oxidase from Streptococcus faecium ATCC 12755.

A procedure was developed to purify the Streptococcus faecium ATCC 12755 L-alpha-glycerophosphate oxidase. The molecular weight of the purified enzyme was 131,000 and the subunit molecular weight was 72,000. Two moles of FAD were bound/mol of enzyme. Apo-L-alpha-glycerophosphate oxidase displayed physical properties similar to the holoenzyme as judged by electrophoresis in 10% buffer gels at pH 8.5 and by centrifugation in a 5 to 20% linear sucrose gradient. The apoenzyme was completely reactivated by incubation with FAD. L-alpha-Glycerophosphate oxidase was specific for L-alpha-glycerophosphate when compared with several other pohsphorylated glycerol and sugar derivatives. Oxygen was the preferred electron acceptor. At 10 mM DL-alpha-glycerophosphate (below the Km of 26 mM for L-alpha-glycerophosphate), activity was increased from 2.6- to 10-fold by increasing the buffer concentration from 0.01 to 0.1 m. This buffer effect was observed with potassium phosphate and other anionic buffers. In 0.001 m potassium phosphate buffer, pH 7.0, activity was increased by several divalent metal ions, including 10 mM CaCl2 (7.7-fold activation) and 10 mM MgCl, (6.8-fold activation). Fructose 6-phosphate and fructose1-phosphate were inhibitors of the L-alpha-glycerophosphate oxidase.     

Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells

Juxtanuclear birefringent caps (FC) containing 10-nm filaments form during the early stages of baby hamster kidney (BHK-21) cell spreading. FC are isolated from spreading cells after replating by treatment with 0.6 M KCl, 1% Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) and DNase I in phosphate-buffered saline. Purified FC are birefringent and retain the pattern of distribution of 10-nm filaments that is seen in situ. Up to 90% of the FC protein is resolved as two polypeptides of approximately 54,000 and 55,000 molecular weight on sodium dodecyl sulfate (SDS) polyacrylamide gels. The protein is immunologically and biochemically distinct from tubulin as determined by indirect immunofluorescence, double immunodiffusion, one-dimensional peptide mapping by limited proteolysis in SDS gels, and amino acid analysis. The BHK-21 FC amino acid composition, however, is very similar to that obtained for 10-nm filament protein derived from other sources including brain and smooth muscle. Partial disassembly of 10-nm filaments has been achieved by treatment of FC with 6 mM sodium- potassium phosphate buffer, pH 7.4. The solubilized components assemble into distinct 10-nm filaments upon the addition of 0.171 M sodium chloride.     

Isolation and characterization of proteoheparan sulfate from plasma membranes of an ascites hepatoma, AH 66

A proteoglycan was isolated from plasma membranes prepared from AH 66 cells by the following procedure. The plasma membranes were isolated from cells according to the method devised by Funakoshi and Yamashina (1976) J. Biochem. 80, 1185-1193), then the membranes were made lipid-free. The lipid-free membranes were solubilized with 5 mM sodium phosphate buffer, pH 7.0, containing 0.5% sodium dodecyl sulfate (SDS), then the solution was fractionated on a Sepharose CL 6B column. The proteoglycan eluted near the void volume fraction was further purified by repeated precipitation with cetylpyridinium chloride (CPC). The proteoglycan isolated was homogeneous on electrophoresis on a cellulose acetate strip and was identified as proteoheparan sulfate. The preparation contained 10.6% protein, its amino acid composition being characterized by high contents of glutamic acid, aspartic acid, proline, glycine, threonine, and serine.     

The interrelation between high- and low-molecular-weight forms of GM1-beta-galactosidase purified from porcine spleen

1) Two forms of acid beta-galactosidase [EC 3.1.23] with different molecular weights catalyzing the hydrolysis of GM1-ganglioside and p-nitrophenyl-beta-D-galactoside were separated and purified from porcine spleen. 2) The apparent molecular weights were 400,000-600,000 and 70,000-74,000 for the high (termed Am form) and low (termed A1 form) molecular weight forms, respectively. 3) On examination by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis, both forms of the enzyme had a common protein band of molecular weight 63,000, and the Am form showed three additional protein bands with molecular weights of 31,000, 21,000, and 20,000. 4) Both forms of the enzyme had similar catalytic functions with regard to pH-optimum, Km, substrate specificity and sensitivity to substrate analogues and other substances such as detergents, bovine serum albumin (BSA) and NaCl. 5) Both forms of the enzyme were fairly stable upon preincubation at 45 degrees C at acidic pH (pH 4.5), but lost their activities at neutral pH (pH 7.0). 6) The A1 form was a monomer at neutral pH (pH 7.0) and formed a dimer at acidic pH (pH 4.5). However, most of the Am form could not be converted to a dimeric form on gel filtration at acidic pH.     

Effect of protein concentration on the molecular weight of delta5-3-ketosteroid isomerase.

The molecular weight of delta-5-3-ketosteroid isomerase from Pseudomonas testosteroni was determined by means of sedimentation equilibrium and exclusion chromatography over a wide range of enzyme concentrations in 0.2 M potassium phosphate buffer, pH 7.0. In addition, the sedimentation constant of the enzyme was determinded over an extended range of concentrations. The enzyme was found to have a molecular weight of 26,000 plus or equal to 1,000, suggesting that it is a dimer of identical or similar 13,400 molecular weight polypeptide chains. In the ultracentrifuge this dimeric species was found to undergo aggregation at enzyme concentrations above 2 mg per ml and dissociation at enzyme concentrations below 0.05 mg per ml. Exclusion chromatography studies indicate that under the conditions of chromatography the oligomeric enzyme is partially dissociated at enzyme concentrations in the range 0.2 to 0.002 mug per ml. These results suggest that under conditions of enzyme assay in 0.2 M potassium phosphate buffer, pH 7.0, isomerase is in a monomeric state of aggregation.