Protein chromatography on adsorbents with hydrophobic and ionic groups. Some properties of N-(3-carboxypropionyl)aminodecyl-sepharose and its interaction with wheat-germ aspartate transcarbamoylase.

1. The charge state of two derivatives of Sepharose prepared by the CNBr activation method were studied by acid-base titration and by ion-exchange chromatography. Dodecyl-Sepharose exhibited cationic groups (21mumol/ml of settled gel; pKa=9.6) that were tentatively assigned to the coupling isourea group. 2. CPAD-Sepharose [N-(3-carboxypropionyl)aminodecyl-Sepharose] has anionic (carboxyl) groups (pKa=4.5) and cationic groups (pKa=9.6) in roughly equal concentrations (e coupling group. CPAD-Sepharose is slightly negatively charged at pH 7.0 and substantially negatively charged at pH 8.5. 3. The pKa values of dodecyl-Sepharose and CPAD-Sepharose are unaffected by a 100-fold increase in the concentration of KCl. 4. CPAD-Sepharose has considerable affinity for wheat-germ aspartate transcarbamoylase at pH 8.5 when the adsorbent and enzyme are both negatively charged. The interaction involves the C10 chain but is relatively moderate compared with C10 chains associated only with positive charge. 5. Desorption of the enzyme adsorbed to CPAD-Sepharose can be achieved by raising the pH to increase the electrostatic repulsion, or by introducing the detergent sodium deoxycholate. Acetone and butan-1-ol also weaken the adsorption at pH 8.5. 6. High concentrations of sodium acetate or sodium phosphate induced the enzyme to bind more tightly to CPAD-Sepharose. 7. These results are discussed in terms of a 'repulsion-controlled' model or hydrophobic chromatography.     

Exhaustive removal of N-glycans from ascorbate oxidase: effect on the enzymatic activity and immunoreactivity

Purified ascorbate oxidase from Cucurbita pepo medullosa hasbeen subjected to enzymatic deglycosylation using peptide N-glycosidaseF. Experimental conditions were chosen to obtain efficientlydeglycosylated and active ascorbate oxidase: in particular,three different detergent solutions were added separately tothe incubation mixtures prior to the peptide N-glycosidase F.The detergent solution made of 0.1% (w/v) sodium dodecyl sulphate+ 0.5% (v/v) Nonidet P-40 proved to be the only one effectivefor our purpose. Our results indicate that: (i) the presenceof detergents did not affect the enzymatic activity; (ii) fullydeglycosylated enzyme retained its activity compared with thenative form. Moreover, anti-native ascorbate oxidase antibodiesscarcely recognized deglycosylated protein. ascorbate oxidase blot deglycosylation     

Purification and characterization of an extracellular polygalacturonase from Lactobacillus plantarum strain BA 11

Lactobacillus plantarum produced extracellular polygalacturonase in a medium containing 1.5% low methyl-pectin (w/v) and 0.5% glucose (w/v) as inducers. The enzyme was purified (approximately 70-fold) by ammonium sulphate fractionation, Sephadex G-100 gel filtration and DEAE-cellulose ion exchange chromatography. Two peaks (PG I and PG II) of enzymic activity were obtained from the DEAE-cellulose column. The molecular mass of PG I was similar to that of PG II (32 000 Da). The K _m values of PG I and PG II for sodium polypectate were calculated to be 1.63 mg/ml and 1.78 mg/ml respectively. Their isoelectric points were about pH 5.5. The pH optimum was 4.5, while the optimum temperature was 35°C for both PG I and PG II. The two purified enzymes had similar endo modes of action on polygalacturonic acid, as determined by comparison of viscosity reduction and reducing group release.     

Isolation and some molecular parameters of elastase-like normal proteinases from horse blood leucocytes.

Cytoplasmic granules were isolated from horse blood polymorphonuclear leucocytes by the heparin method and extracted with 0.9% NaCl by repeated freezing. Soluble proteins were separated on a column of Sephadex G-75 followed by chromatography on a column of CM-Sephadex with a NaCl gradient. Gel filtration, density-gradient centrifugation, isoelectric focusing and 0.1% sodium dodecyl sulphate/polyacrylamide-gel electrophoresis at pH 7.0 and at pH 4.5 were used to determine molecular parameters of proteinases. Three enzymes hydrolysing both casein and N-benzyloxycarbonyl-L-alanine nitrophenyl ester were found in the granule extract: proteinase 1, mol.wt. 38000, pI5.3; proteinase 2A, mol.wt. 24500, pI8.8; and proteinase 2B, mol.wt. 20500, pI above 10. The latter two elastase-like proteinases were purified to apparent homogeneity.     

Purification and properties of arylsulphatase A from rabbit testis.

Rabbit testis arylsulphatase A was purified 140-fold with a recovery of 20% from detergent extracts of an acetone-dried powder by using DE-52 cellulose column chromatography, gel filtration on Sephadex G-200 and preparative isoelectric focusing. The purified enzyme showed one major band with one minor contaminant on electrophoresis in a 7.5% (w/v) polyacrylamide gel at pH8.3. On sodiumdodecyl sulphate/polyacrylamidegel electrophoresis, a single major band was observed with minor contaminants. The final preparation of enzyme was free from general proteolytic, esterase, hyaluronidase, beta-glucuronidase and beta-galactosidase activities. Rabbit testicular arylsulphatase A exists as a dimer of mol.wt. 110000 at pH7.1. At pH5.0 the enzyme is a tetramer of mol.wt. 220000. Arylsulphatase A appears to consist of two identical subunits of mol.wt. 55000 each. The highly purified enzyme has pI4.6. The enzyme hydrolyses p-nitrocatechol sulphate with Km and Vmax, of 4.1 mM and 80nmol/min respectively, but has no activity toward p-nitrophenyl sulphate. The pH optimum of the enzyme varies with the incubation time. By applying Sephacex G-200 chromatography and preparative isoelectric focusing, one form of enzyme was obtained. The enzyme has properites common to arylsulphatase A of other sources with respect to the anomalous time-activity relationship, pI, inhibition by PO42-, SO32- and Ag+ ions and substrate affinity to p-nitrocatechol sulphate. However, the enzyme shows the temperature optimum of arylsulphatase B of other species.     

The adsorption of proteins and protein--dodecyl sulphate complexes on N-(3-carboxypropionyl)aminodecyl-Sepharose.

Equilibrium and kinetic aspects of the binding of several proteins to N-(3-carboxypropionyl)aminodecyl-Sepharose, an amphiphilic ampholytic adsorbent, were studied at 22 degrees C, pH 7.0, I 0.10--0.12. In the absence of detergents Scatchard plots are linear for human haemoglobin and soya-bean trypsin inhibitor, but non-linear for bovine serum albumin, which is also adsorbed more tightly than the other two proteins. The introducion of 3.5mM-sodium dodecyl sulphate causes dramatic increases in the amounts and affinities of serum albumin and haemoglobin adsorbed, but has relatively little effect on the trypsin inhibitor. At concentrations of sodium dodecyl sulphate greater than about 10mM there is a fall in the binding of all proteins, owing to competition from the detergent for binding sites on the adsorbent, and a tendency towards more uniform behaviour by different proteins. Kinetic experiments suggest that in the absence of the detergent haemoglobin and serum albumin are adsorbed initially by mainly ionic forces, but that subsequently hydrophobic forces become dominant. Addition of 3.5 mM-sodium dodecyl sulphate causes pronounced changes in the time course of adsorption of haemoglobin and serum albumin, the nature of the changes being different for each protein. The significance of these results is discussed.     

Chemical and physical properties of the human urinary glycoprotein with gastric antisecretory activity.

The chemical and physical properties of the high-molecular-weight glycoprotein (SO20, w = 8S; Ve=Vo on Sephadex G-200) with gastric antisecretory activity extracted from the urine of pregnant women were studied. Gel filtration in the presence of sodium dodecyl sulphate and sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis indicated subunit mol.wts. of 16 000 +/- 1500 and 13 000 +/- 1000 respectively. Reaggregation of the subunits and partial recovery of the biological activity were observed on removal of the detergent. The partial C-terminal sequence was found to be Phe-Tyr-Leu-Val-OH, whereas glycine appears to be the N-terminal amino acid. The carbohydrate composition was examined; all galactosamine was found to be O-glycosidically linked to the polypeptide chain.     

Towards crystallization of hydrophobic myelin glycoproteins: P0 and PASII/PMP22

The preparation of a pure and homogeneous protein sample at proper concentration is a prerequisite for success when attempting their crystallization for structural determination. The detergents suitable for solubilization particularly of membrane proteins are not always the best for crystallization. Myelin of the peripheral nervous system of vertebrates is the example of a membrane for which neutral or "gentle" detergents are not even strong enough to solubilize its proteins. In contrast, sodium- or lithium-dodecyl sulfate is very effective. We solubilized myelin membrane in 2%(w/v) sodium dodecyl sulfate, followed by chromatographic purification of the hydrophobic myelin glycoproteins P0 and PASII/PMP22, and finally, we have exchanged the sodium dodecyl sulfate bound to protein for other neutral detergents using ceramic hydroxyapatite column. Theoretically, we should easily exchange sodium dodecyl sulfate for any neutral detergent, but for some of them, the solubility of myelin glycoproteins is low. To monitor the potential variability in the secondary structure of glycoproteins, we have used circular dichroism. Sodium dodecyl sulfate seems to be the appropriate detergent for the purpose of purification of very hydrophobic glycoproteins, since it can be easily exchanged for another neutral detergent.     

Association of myelin basic protein with detergent micelles.

Equilibrium measurements of the binding of central nervous system myelin basic protein to sodium dodecyl sulphate, sodium deoxycholate and lysophosphatidylcholine have been obtained by gel permeation chromatography and dialysis. This protein associates with large amounts of each of these surfactants: the apparent saturation weight ratios (surfactant/protein) being 3.58 +/- 0.12 and 2.30 +/- 0.15 for dodecyl sulphate at ionic strengths 0.30 and 0.10, respectively 1.34 +/- 0.10 for deoxycholate (at 0.12 ionic strength) and 4.0 +/- 0.5 for lysophosphatidylcholine. Binding to the ionic surfactants increases markedly close to their critical micelle concentrations. Sedimentation analysis shows that at 0.30 ionic strenght in excess dodecyl sulphate the protein is monomeric. It becomes dimeric when the binding ratio falls below 1 at a free detergent concentration of approximately 0.25 mM: below this concentration much of the protein and deterent forms an insoluble complex. The amount of dodecyl sulphate bound at high concentrations and at both above-mentioned ionic strengths corresponds closely to that expected for interaction of a single poly-peptide with two micelles. Variability of deoxycholate micelle size on interaction with other molecules precludes a similar analysis for this surfactant. Association was observed only with single micelles of lysophosphatidylcholine. The results provide strong evidence for dual lipid-binding sites on basic protein and indicate that lipid bilayer cross-linking by this protein may be effected by single molecules.     

A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase

Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37 °C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.     

Nuclear proteins. VI. Fractionation of chromosomal non-histone proteins using hydrophobic chromatography.

Mouse liver non-histone proteins, isolated by hydroxyapatite chromatography, were fractionated by hydrophobic chromatography using omega-amino-decyl-agarose omega-amino butyl-agarose, decyl-agarose, butyl-agarose, phenyl-Sepharose, and CPAD-Sepharose. Two column loading techniques were used. In the 0.35 M NaCl technique, the proteins were dialized into 0.35 M NaCl, applied to the column and initially eluted with 0.35 M NaCl. In the 40% (NH4)2SO4 technique, the non-histone proteins were mixed with the hydrophobic agarose, dialized against 40% (NH4)2SO4, and initially eluted with 40% (NH4)2SO4. In both cases the columns were subsequently eluted with 10 mM Tris-HCl, pH 7.5, 0.35, 1.0 and 5.0 M LiBr, and finally with 1% sodium dodecyl sulfate. The 0.35 M NaCl technique, using decyl-agarose and phenyl-Sepharose, resulted in a single step marked enrichment of the major hnRNA proteins (1 M LiBr fraction). The 40% (NH4)2SO4 technique resulted in a single step isolation of a pair of 15-20 000 dalton polypeptides.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens.

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

The preparation of calmodulins from barley (Hordeum sp.) and basidiomycete fungi.

1. Calmodulin-like proteins were purified from the fruiting bodies of higher (basidiomycete) fungi and barley (Hordeum sp.) shoots. 2. These calmodulins have electrophoretic mobilities on 10% (w/v) polyacrylamide gels at pH 8.3 in the presence of 6 M-urea and at pH 8.3 in the presence of 0.1% sodium dodecyl sulphate similar to that of bovine brain calmodulin. They interacted with rabbit skeletal-muscle troponin I in the presence of Ca2+. 3. Barley and fungal calmodulins activated myosin light-chain kinase and phosphodiesterase in the presence of Ca2+, although the amounts needed were at least an order of magnitude greater than is required to produce the same effect with mammalian calmodulin. 4. Amino acid analyses indicated a number of differences from the mammalian protein, most notably the absence of trimethyl-lysine. 5. By using 125I-labelled calmodulin, a small amount of calmodulin-binding protein was detected in homogenates of barley and fungi. 6. No protein corresponding to calmodulin could be found in Escherichia coli or yeast, although a relatively high concentration of a protein that bound calmodulin was detected in E. coli by this technique.     

Affinity purification and some molecular properties of human liver alkaline phosphatase.

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.     

A simple method for purification of epoxide hydratase from rat liver.

Rat liver epoxide hydratase was purified 460-fold to homogeneity by detergent solubilization and ion-exchange chromatography. The enzyme obtained in high yield (36%) exhibited a specific activity of 479nmol of styrene glycol formed/min per mg of protein, with styrene oxide as substrate. Only one polypeptide-staining band, mol.wt. 49500, was visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.     

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.     

Direct quantification of mitochondrial DNA and its 4.9-kb common deletion without DNA purification

We studied the extraction and analysis of integral membrane proteins possessing hydrophobic and hydrophilic domains and found that a nonionic detergent called MEGA-10, used in lysis buffers, had a superior extraction effect compared to most conventional detergents. A sodium dodecyl sulfate (SDS) concentration of >0.4% (w/v) in the sample buffer was crucial for those proteins to be clearly analyzed by electrophoresis and Western blotting. Furthermore, MEGA-10 had the tendency to maximally extract proteins around its critical micelle concentration (CMC) of 0.24% (w/v). These solutions can greatly assist functional investigations of membrane proteins in the proteomics era.     

Studies on the ribonucleic acids of fresh and processed tea leaves

1. A marked decrease in the total RNA content during the withering process of tea leaves was found. During the fermentation process, there was a small but significant decrease in the total RNA content. 2. During isolation of RNA from tea leaf tissues, the action of leaf ribonuclease was minimized by the addition of sodium dodecyl sulphate during extraction; 1% (w/v) sodium dodecyl sulphate in 0.2m-tris-hydrochloric acid buffer, pH8.0, containing 0.005% EDTA was found to be most efficient for the extraction and gave about 93% yield. 3. The total RNA preparations isolated from fresh, withered and fermented tea leaves were compared with regard to nucleotide composition and spectral characteristics. The total RNA preparations from all three sources contained more purines than pyrimidines (purine/pyrimidine ratio 1.47-1.52).     

Purification and characterization of rat adipose tissue lipoprotein lipase.

Lipoprotein lipase (EC 3.1.1.34) extracted from adipose tissue of glucose-fed rats with 5 mM-sodium barbital, pH 7.5, containing 20% (v/v) glycerol and 0.1% (v/v) Triton X-100, was partially purified by affinity chromatography on heparin linked to Sepharose 4B. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the partially purified enzyme preparation revealed the presence of two major Coomassie-staining bands (mol.wts. 62 000 and 56 000) as well as a number of minor bands. Treatment of partially purified enzyme with [1,3-3H]di-isopropyl fluorophosphate resulted in the incorporation of radiolabel into the band of mol.wt. 56 000, but not into the band of mol.wt. 62 000. Both the amount of the 56 000-mol.wt. polypeptide and the incorporation of [1,3-3H]di-isopropyl fluorophosphate into this band were greatly reduced in the enzyme preparations isolated from adipose tissue of 48 h-starved rats. whereas the amount of the 62 000-mol.wt. polypeptide was unaffected by starvation. Purification of lipoprotein lipase from adipose tissue of glucose-fed rats was also carried out using affinity chromatography on Sepharose 4B linked to heparin with low affinity for antithrombin-III. This procedure resulted in the presence of a single band of mol.wt. 56 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These results suggest that the polypeptide of mol.wt. 56 000 corresponds to the subunit of lipoprotein lipase, whereas the 62 000-mol.wt. polypeptide probably represents antithrombin-III.     

Size-exclusion chromatographic study of the reduction of recombinant hepatitis B surface antigen

The reduction of the P. pastoris-derived hepatitis B surface antigen (HBsAg) has been investigated by size exclusion chromatography performed in a detergent solution containing 0.3% sodium dodecyl sulfate (SDS) and 0.1 M Tris–HCl, pH 7.0. The HBsAg, reduced under different conditions and passed through the TSK G4000 SW column (600×7.5 mm I.D.) at 0.9 ml min⁻¹, was resolved into two peaks corresponding to the reduced, monomeric, and non-reduced forms, respectively. Under these conditions, the antigen fraction corresponding to the HBsAg dimer can be separated and completely reduced to monomers by repeated reductive treatment with simultaneous lipid removal. The efficiency of reduction was maximal after sample treatment with an equal volume of a solution containing 417 mM dithiothreitol, 4.2% (w/v) SDS and 16% (v/v) 2-mercaptoethanol. In conclusion, complete reduction of recombinant HBsAg to monomer subunits is possible and depends on the efficiency of lipid removal during the reductive treatment.