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.     

Determination of profiles of non-collagenous proteins from rat bones by sodium dodecyl sulfate high-performance liquid chromatography.

Sodium dodecyl sulfate-high-performance liquid chromatographic (SDS-HPLC) techniques for screening profiles of bone non-collagenous proteins (NCP) are described and compared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques. NCPs were obtained from long bones of neonatal and mature rats by sequential extraction with 4 M guanidine hydrochloride (GdnHCl) and 0.25 M EDTA followed by desalting. Desalted extracts were subjected to SDS-PAGE and SDS-HPLC. The results of the two analyses were comparable. There were differences in NCP profiles between mature rats and pups and between the GdnHCl and EDTA extracts. The methods described can be used for qualitative comparison of treatments and as a basis for further study.     

Rapid Purification of the Unstable Enzyme Carbamoyl Phosphate Synthase by High Pressure Liquid Chromatography

Abstract

Extracts of soluble proteins obtained from rat liver mitochondria by freeze-thawing and subsequent diafiltration were fractionated by HPLC on a I 250 protein column. The column was eluted either with 0.05 M phosphate buffer pH 6.85 or 0.1 M acetate buffer pH 7.15. Specific fractions obtained by elution with either phosphate or acetate buffer showed a 6.1-fold or 5.5-fold increase in the specific activity of Carbamoyl phosphate synthase when compared with that of crude mitochondrial preparations. The purification and the molecular weight of carbamoyl phosphate synthase were verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.     

Viscosities and apparent molar volumes of some amino acids in water and in 6M guanidine hydrochloride at 25°C

Apparent molar volumes and viscosity B- and D- coefficients of amino acids glycine, valine, proline, serine and arginine have been determined in water and in aqueous 6M guanidine hydrochloride (GuHCl) solution at 25°C. Transfer volumes and transfer viscosity B-coefficients were evaluated for the amino acids studied in going from water to 6M GuHCl. These transfer properties which were all positive were interpreted in terms of strong interactions of GuHCl molecules with the charged centers of amino acid molecules. A comparison of results obtained in this work for GuHCl and those obtained from literature for urea has shown that GuHCl has stronger interactions than urea with amino acids. This finding explained the previous experimental observations on GuHCl being a stronger denaturing agent than urea for proteins.     

High-performance hydroxyapatite chromatography of integral membrane proteins and water-soluble proteins in complex with sodium dodecyl sulphate.

Integral membrane proteins from human erythrocytes were fractionated in the presence of sodium dodecyl sulphate (SDS) on four types of high-performance hydroxyapatite columns. A column of 2-microns sintered hydroxyapatite beads from Asahi Optical (Tokyo, Japan) gave the best resolution. With this column, glycophorin was eluted early in a gradient of increasing sodium phosphate buffer concentration, the glucose transporter was eluted later in two zones, one of which contained this protein alone, and the anion transporter was eluted last. Water-soluble proteins applied in complex with SDS also separated reasonably well upon elution. The water-soluble proteins and the membrane proteins were all eluted mainly in the order of increasing polypeptide length, but with considerable individual variation. SDS-polypeptide complexes are probably adsorbed onto hydroxyapatite by the interaction of positively charged amino acid side groups with phosphate ions (at P-sites) and of negatively charged amino acid side groups and polypeptide-bound dodecyl sulphate anions with calcium ions (at C-sites). As a rule, the number of charged side groups and dodecyl sulphate anions, and thus the number of binding sites, increases with the polypeptide chain length, which explains the general order of release of the polypeptides.     

Resolution and quantitation of apolipoproteins A-I and A-II from human high-density lipoprotein by size exclusion high-performance liquid chromatography

Apolipoproteins A-I and A-II, extracted from human high-density lipoprotein (HDL), were resolved and quantified by size exclusion high-performance liquid chromatography on TSK 125 and TSK 250 analytical columns connected in series without the use of chemical denaturants or detergents in the eluent buffer. The columns were pre-equilibrated with a solution containing 0.1 M sodium phosphate, pH 7.2, 0.2 M sodium chloride at a flow-rate of 1 ml/min. Delipidated HDL (1 mg protein per ml) was resolved into two populations of apolipoprotein (apo) A-I: one representing the apo A-I monomer and the other, a self-associated form with a molecular weight of approximately 120,000 daltons. The column eluates were screened for immunoreactivity to apo A peptides, and the identity of each peak was confirmed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis followed by immunoblot analysis. Apo A-I peptides isolated by high-performance liquid chromatography disrupted unilamellar phospholipid vesicles to form smaller phospholipid particles that eluted on gel filtration columns within the size range of HDL. Thus, a rapid method for the isolation and quantitation of non-denatured apolipoproteins from HDL has been developed using size exclusion high-performance liquid chromatography.     

Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using Tris‐acetate polyacrylamide gels

To separate and analyze giant and small proteins in the same electrophoresis gel, we have used a 3–15% polyacrylamide gradient gel containing 2.6% of the crosslinker bisacrylamide and 0.2 M of Tris‐acetate buffer (pH 7.0). Samples were prepared in a sample buffer containing lithium dodecyl sulphate and were run in the gel described above using Tris‐Tricine‐SDS‐sodium bisulfite buffer, pH 8.2, as electrophoresis buffer. Here, we show that this system can be successfully used for general applications of SDS‐PAGE such as CBB staining and immunoblot. Thus, by using Tris‐acetate 3–15% polyacrylamide gels, it is possible to simultaneously analyze proteins, in the mass range of 10–500 kDa, such as HERC1 (532 kDa), HERC2 (528 kDa), mTOR (289 kDa), Clathrin heavy chain (192 kDa), RSK (90 kDa), S6K (70 kDa), β‐actin (42 kDa), Ran (24 kDa) and LC3 (18 kDa). This system is highly sensitive since it allows detection from as low as 10 μg of total protein per lane. Moreover, it has a good resolution, low cost, high reproducibility and allows for analysis of proteins in a wide range of weights within a short period of time. All these features together with the use of a standard electrophoresis apparatus make the Tris‐acetate‐PAGE system a very helpful tool for protein analysis.     

Secondary structural changes of chymotrypsinogen A,alpha-chymotrypsin,and the isolated polypeptides Cys1-Leu13, Ile16-Tyr146, and Ala149-Asn245 in sodium dodecyl sulfate,urea and guanidine hydrochloride

Secondary structural changes of chymotrypsinogen A,-chymotrypsin, and their isolated polypeptides Cys¹-Leu¹³, Ile¹⁶-Tyr¹⁴⁶, and Ala¹⁴⁹-Asn²⁴⁵were examined in aqueous solutions of sodium dodecyl sulfate (SDS), urea, and guanidine hydrochloride (residue numbers from chymotrypsinogen). After the fragmentation by the cleavage of disulfide bridges in-chymotrypsin, the helical structure was formed in the isolated polypeptide 16–146 where the helical segments do not exist in the protein state. The polypeptide 149–245, where the helical segments of the parent protein are originally located, contained no helices. The polypeptide 1–13 was almost disordered. The three polypeptides, chymotrypsinogen,-chymotrypsin and the polypeptide 16–146, clearly showed differences in the stabilities of helical structures in solutions of urea and guanidine hydrochloride. The addition of SDS accelerated the formation of helical structures in each polypeptide except for 1–13.     

Comparative study for separation of aquatic humic substances by capillary zone electrophoresis using uncoated, polymer coated and gel-filled capillaries

Several comparative capillary zone electrophoresis (CZE) experiments were carried out by means of uncoated, polyvinyl alcohol (PVA) and polyacrylamide (PAA) coated silica open tubular capillaries and gel-filled capillaries (linear non-cross-linked polyacrylamide, PAGE, by a pre-coated PAA capillary) using different kinds of background electrolytes (BGEs) and organic modifiers for characterization of aquatic dissolved humic matter (DHM). Organic compounds, such as acetic acid, acetate buffer, methanol, ethylene glycol, acetonitrile, dimethylsulphoxide, 5 M urea and sodium dodecyl sulphate (SDS) were tested as sample modifiers to improve the separative power. The fractionation mode by a PVA coated open tubular capillary using 40 mM phosphate buffer at pH 6.8 and 5 M urea-water as the sample modifier turned out to be fairly practical as well as its PAA homologue. Linear non-cross-linked PAGE with 10% gel concentration and 5 M urea-water as the sample modifier using 40 mM phosphate buffer at pH 6.8 produced the most reliable results as to the adaptation of physical gels, especially if the interactions of humic solutes with the gel matrix are not critical. The addition of SDS in the linear PAGE gel increased the interaction of humic solutes with the gel matrix but also improved the separative power and strengthened the chaotropic effect of the urea modifier.     

Per(6-guanidino-6-deoxy)cyclodextrins: synthesis, characterisation and binding behaviour toward selected small molecules and DNA

Per(6-guanidino-6-deoxy)-cyclodextrins , and are novel derivatives, resulting from homogeneous introduction of the guanidino group at the primary side of alpha-, beta- and gamma-cyclodextrins. The products were obtained from the corresponding amino derivatives, as direct guanidinylation of the known bromo-cyclodextrins provided mixtures. The new compounds were fully characterized by NMR spectroscopy and other analytical methods, and their interaction with guest molecules was studied. Strong complexation with 4-nitrophenyl phosphate () disodium salt was observed (K(binding) approximately 5 x 10(4) M(-1)), whereas the non-phosphorylated substrate nitrobenzene () formed a very weak complex. 2D ROESY spectra revealed cavity inclusion in both cases, however the orientation of was opposite to that of , such that the phosphate group is oriented toward the primary side facing the guanidine groups. The strong affinity of towards the phosphorylated guest suggested that interaction with DNA was possible. The new compounds were found to completely inhibit the migration of ultra pure calf thymus DNA during agarose gel electrophoresis, whereas no effects were observed with guanidine alone or with the plain cyclodextrins. Further, the condensation of DNA into nanoparticles in the presence of was demonstrated by atomic force microscopy, confirming strong electrostatic interaction between the biopolymer and the multicationic products . The strong guanidine-phosphate interactions between and DNA were therefore attributed to the clustering of the guanidine groups in the primary area of the cyclodextrin. Cavity effects could not be assessed.     

Capillary electrophoresis of seed 2S albumins from Lupinus species

Two modes of capillary electrophoresis (CE)--free-solution capillary zone electrophoresis (CZE) and sodium dodecyl sulfate capillary electrophoresis (SDS-CE) using a non-gel sieving matrix--have been developed for comparative analysis of low-molecular-mass 2S albumin isoforms from lupins. The albumin fraction and 2S albumins were separated in uncoated fused-silica capillary by CZE with 0.02 M phosphate buffer, pH 7.3, containing the sodium salt of phytic acid. The use of phytic acid (0.025 M) as buffer modifier and ion-pairing agent improved migration reproducibility, peak shape and separation efficiency. The reduced 2S albumins were separated by SDS-CE using a high concentration (0.3-0.5 M) mixture of tris(hydroxymethyl)aminomethane and borate buffers in uncoated fused-silica capillary. Of the various polymers used as non-gel sieving matrix, SDS-CE with a 10% dextran solution was found to be suitable for separation of 2S albumin polypeptides with molecular masses of 4,000-7,000 and 8,000-11,000. The addition of glycerol or ethylene glycol to the SDS separating buffer improved the resolution of polypeptides. The examined Lupinus species showed species-specific CZE and SDS-CE migration profiles of the 2S albumins.     

Self-assembling of guanidine-type surfactant

Self-assembling characteristics of dodecylguanidine hydrochloride (C 12G), a cationic surfactant with a guanidine group in its molecule, were investigated and compared with those of dodecyltrimethylammonium chloride (DTAC) and sodium dodecylsulfate (SDS). Introduction of a guanidine group into the surfactant molecule was found to increase its assembly formability more than that of the trimethylammonium group on the basis of the experimental results on the phase diagram, Kraft point, area occupied per molecule at the air-water interface, and micellar aggregation number of C 12G. Thermodynamic parameters for micelle formation suggested that an attractive force acts between guanidine groups of C 12G molecules to facilitate their assembly formation. The presence of this force was evidenced by changes in the (1)H NMR and IR spectra before and after micelle formation of the guanidine-type (G-type) surfactant, indicating that the increased assembly formability is caused by an increase in hydrogen bonding between guanidine groups of the surfactant via water molecules.     

Effect of Sodium Dodecyl Sulfate on Concanavalin A-Sepharose During Affinity Chromatography of High-Affinity Binding Toxin Protein Of Bacillus thuringiensis subsp. kurstaki

Abstract

Concanavalin A- Sepharose affinity chromatography is a powerful tool for isolation or purification of peripheral or integral membrane proteins or other glycoproteins. The insecticidal crystal toxin from Bacillus thuringiensis subsp. kurstaki is a glycoprotein containing “high-mannose” or “hybrid”-type sugar chains. The protein has a high binding affinity for concanavalin A lectin and could not be eluted even with 0.5M methyl α-D-mannopyranoside. Nonspecific elution with 0.03% SDS coeluted the matrix con A with bound protein. Experimental results indicated that con A leaching is mainly because of inclusion of detergents in buffer systems and may not be directly related to the nature of the sample protein.

² Abbreviations used: Con A: concanavalin A, SDS: sodium dodecyl sulfate, SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MeG: methyl α-D-glucopyranoside, MeM: methyl α-D-mannopyranoside     

Identification of gel-separated tumor marker proteins by mass spectrometry

Two-dimensional gel electrophoresis with subsequent analysis by mass spectrometry was applied to study differences in protein expression between benign and malignant solid tumors from human beast, lung and ovary cells. Cells from freshly resected clinical material were lysed and the extracts were subjected to isoelectric focusing with immobilized pH gradients followed by second-dimensional separation on 10-13% sodium dodecyl sulfate (SDS)/polyacrylamide gels. Polypeptides were identified using matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry after in-gel protein digestion. Some of the upregulated polypeptides in malignant cells are of potential importance as markers of tumor proliferation. Twenty such proteins were identified, ten constituting novel identifications and ten sequence verifications of previously gel-matched proteins. The proteins identified span a wide range of functions, but several cases of protein truncation were found. Truncated forms of cytokeratins 6D and 8, and of cathepsin D were identified. Truncated froms of these over-expressed proteins support the presence of proteolytic processing steps in tumor material. The protein processing and the difference between protein and mRNA abundancies in tumors of different malignancy and origin suggest that studies at the protein level are important for an understanding of tumor phenotypes.     

Effect of strong detergents and chaotropes on the detection of proteins in two-dimensional gels.

The solubilization of a particular protein is mandatory for its subsequent resolution and detection in two-dimensional gels. However, the extraction solutions, that are compatible with the first-dimensional separation step, such as urea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), do not solubilize all proteins in a sample. We studied the effect of various common, strong detergents and chaotropes, widely used as solubilizing agents, such as sodium dodecyl sulfate, lithium dodecyl sulfate and guanidine hydrochloride, on the solubilization of the total and membrane proteins of the bacterium Haemophilus influenzae. The proteins solubilized with each system were analyzed by two-dimensional electrophoresis and these of interest were identified by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Use of sodium dodecyl sulfate, lithium dodecyl sulfate or guanidine hydrochloride for the solubilization of total proteins of the microorganism resulted in the detection of several additional spots, representing mainly outer membrane proteins, in comparison with those detected in the soluble protein fraction. Solubilization of the proteins of the cell envelope fraction with sodium dodecyl sulfate did not result in a more efficient protein detection when compared to the extraction with the urea/CHAPS system. When the dry immobilized pH gradient strips were rehydrated in a solution containing the proteins of the membrane fraction solubilized with sodium dodecyl sulfate or lithium dodecyl sulfate, a larger number of protein spots were detected in comparison with strips that were rehydrated in the urea/CHAPS solution. However, no improvement was observed in comparison with protein application in sample cups. The additional proteins detected with the use of strong detergents and chaotropes are in the majority difficult to solubilize and less hydrophobic proteins.     

纯化基因重组白细胞介素-2的初步研究

 采用羟基磷灰石 (HAP)作为填料 ,以pH 6 8的磷酸盐缓冲液为流动相 ,用制备型高效液相色谱来分离提纯基因重组白细胞介素 2 (IL 2 ) ,通过SDS 聚丙烯酰胺凝胶 (SDS PAGE)电泳测定IL 2的纯度 ,测定其活性为1× 1 0 6 U/mg。该法对IL 2的纯化、含量测定及活性测定等效果良好。     

Micellar‐Enhanced Spectrofluorimetric Determination of Trazodone Hydrochloride in Human Urine and Serum

Abstract

A novel developed spectrofluorimetric method for the determination of trazodone hydrochloride in the presence of sodium dodecyl sulfate (SDS) surfactant micelles was described. Under optimal conditions, there was a good linear relationship between fluorescence intensity and trazodone hydrochloride concentration in the range of 4.0×10^?9 to 8.0×10^?6 mol · l^?1with the detection limit of 1.3×10^?9 mol · l^?1 (S/N=3). This method has been used to determine trazodone hydrochloride in biological fluids.     

Positive cooperative mechanistic binding of proteins at low concentrations: a comparison of poly (sodium N-undecanoyl sulfate) and sodium dodecyl sulfate

The interactions of the negatively charged achiral molecular micelle, poly (sodium N-undecanoyl sulfate) (poly-SUS), with four different proteins using intrinsic and extrinsic fluorescence spectroscopic probes, are studied. A comparison of poly-SUS with the conventional surfactant, sodium dodecyl sulfate (SDS), and the monomeric species, SUS, is also reported. In this work, we observed that poly-SUS preferentially binds to acidic proteins, exhibiting positive cooperativity at concentrations less than 1 mM for all proteins studied. Moreover, it appears that the hydrophobic microdomain formed through polymerization of the terminal vinyl group of the monomer, SUS, is largely responsible for the superior binding capacity of poly-SUS. From these results, we conclude that the interactions of poly-SUS with the acidic proteins are predominantly hydrophobic and postulate that poly-SUS would produce superior interactions relative to SDS at low concentrations in polyacrylamide gel electrophoresis (PAGE). As predicted, use of poly-SUS allowed separation of the His-tagged tumor suppressor protein, p53, at sample buffer concentrations as low as 0.08% w/v (2.9 mM), which is 24 times lower than required for SDS in the standard reducing PAGE protocol. This work highlights the use of poly-SUS as an effective surfactant in 1D biochemical analysis.     

Electrophoretic analysis of snake venoms.

Electrophoretic analyses were conducted on snake venoms from 21 species representing Elapidae, Crotalidae and Viperidae. Denatured and native venoms were analyzed by polyacrylamide gel electrophoretic (PAGE) methods with sodium dodecyl sulfate (SDS) and without SDS. Both SDS-PAGE and PAGE profiles of venoms from different snake species indicate that some proteins and polypeptide components of these venoms have common electrophoretic characteristics suggesting a genetic relationship. Conversely, the electropherograms also showed the characteristic protein and polypeptide profiles that could differentiate one snake species from another. Therefore, both SDS-PAGE and PAGE profiles suggest that proteins and polypeptides with similar characteristics abound among subspecies or related species, although each venom has a unique profile that differentiates one species from the other.     

A rapid derivative spectrophotometric method for simultaneous determination of naphazoline and antazoline in eye drops

A zero-crossing first-derivative spectrophotometric method is applied for the simultaneous determination of naphazoline hydrochloride and antazoline phosphate in eye drops. The measurements were carried out at wavelengths of 225 and 252 nm for naphazoline hydrochloride and antazoline phosphate, respectively. The method was found to be linear (r2>0.999) in the range of 0.2-1 microg/ml for naphazoline hydrochloride in the presence of 5 microg/ml antazoline phosphate at 225 nm. The same linear correlation (r2>0.999) was obtained in the range of 1-10 microg/ml of antazoline phosphate in the presence of 0.5 microg/ml of naphazoline hydrochloride at 252 nm. The limit of determination was 0.2 microg/ml and 1 microg/ml for naphazoline hydrochloride and antazoline phosphate, respectively. The method was successfully used for simultaneous analysis of naphazoline hydrochloride and antazoline phosphate in eye drops without any interference from excipients and prior separation before analysis.