Isoelectric protein purification by orthogonally coupled hydraulic and electric transports in a segmented immobilized pH gradient

A new method is described for preparative protein purification, based on isoelectric focusing on immobilized pH gradients. The principle is entirely new, as it is based on keeping the protein of interest isoelectric, in a flow-chamber, and focusing the impurities in the Immobiline gel. For this, a hydraulic flow is coupled orthogonally to an electric flow, sweeping away the non-isoelectric impurities from the recycling chamber. The sample flow-chamber is built in the centre of the apparatus, and is coupled to an upper and lower segment of an immobilized pH gradient. The protein to be purified is kept isoelectric in the flow-chamber and prevented from leaving it by arranging for the extremities of the immobilized pH gradient, forming the ceiling and the floor of this chamber, to have isoelectric points just higher (e.g. +0.05 pH units, on the cathodic side) and just lower (e.g. -0.05 pH units, on the anodic side) than the known pI of the species of interest. Macromolecules and small ions leave the flow chamber at a rate corresponding to a first order reaction kinetics (the plot of log C vs. time being linear). In general, for macromolecules, 12 h of recycling under current allow removal of 95% impurities. After 24 h of recycling, the protein of interest is more than 99.5% pure. The recoveries are very high (approaching 100%) as the sample under purification never enters the Immobiline gel and thus does not have to be extracted from a hydrophilic matrix, as typical of preparative gel electrophoresis.     

Protein desalting by isoelectric focusing in a segmented immobilized pH gradient

We have recently described an apparatus for protein purification based on a segmented Immobiline gel, having one or more liquid interlayers in between. The principle is entirely new, as it is based on keeping the protein of interest isoelectric, in a flow chamber, and focusing the impurities in an Immobiline gel. For this, a hydraulic flow is coupled orthogonally to an electric flow, sweeping away the non-isoelectric impurities from the recycling chamber. We now demonstrate that the present apparatus can be efficiently used for protein desalting. Hemoglobin A samples, containing 50 mM NaCl or 50 mM ammonium acetate, could be efficiently desalted in 2 h of recycling, after which the total salt content had decreased to less than 0.005 mM (a salt decrement of more than 10,000 fold the initial input). However, with polyprotic buffers (sulphate, citrate, phosphate, oligoamines) the desalting process was much slower, typically of the order of 20 h, possibly due to interaction of these species with the surrounding Immobiline matrix. In this last case, outside pH control (e.g. with a pH-stat) is necessary during protein purification, as, due to the faster removal of the monovalent counterion, the solution in the recycling chamber can become rather acidic or alkaline. It is demonstrated that the 2 extremities of the Immobiline segments facing the sample recycling chamber act indeed as isoelectric membranes, having a good buffering capacity, preventing the protein macroion from leaving the chamber by continuously titrating it to its isoelectric point.     

A horizontal apparatus for isoelectric protein purification in a segmented immobilized pH gradient

A modification of the previously described apparatus (Faupel et al. (1987) J. Biochem. Biophys. Methods 15, 147-162), for recycling isoelectric focusing in a segmented immobilized pH gradient, is here reported. The most important improvements are: (1) a horizontal, vs. the previously vertical assembly; (2) a reduction of the thickness of the central flow chamber to 6 mm, vs. the previous 3 cm length and (3) the introduction, at both gel extremities of each Immobiline segment, of polypropylene filters, thus efficiently blocking the gel in situ. The advantages are: (i) the spontaneous removal of air bubbles, which in the vertical apparatus tend to accumulate in the ceiling of the flow chamber and to obstruct the flow of electric current; (ii) a more efficient hydraulic flow with a reduced chance of heating the liquid stream in the flow chamber, due to its reduced length along the separation path and (iii) a reduced risk of gel detachment from the tube walls, due to osmotic swelling caused by focused protein zones in the gel phase and by the fixed Immobiline charges in the polyacrylamide matrix.     

Immobilized pH gradients for isoelectric focusing. III. Preparative separations in highly diluted gels

A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135–172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5%T gels an upper load limit of 40–45 mg protein/ml gel volume is found, in 2.5%T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to ba carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a %T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37°C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5%T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.     

Isoelectric focusing in immobilized pH gradients in the pH 10-11 range

With the synthesis of a new, strongly basic Immobiline (pK 10.3 at 10 degrees C) it has been possible to formulate a new pH 10-11 recipe for focusing very alkaline proteins, not amenable to fractionation with conventional isoelectric focusing in carrier ampholyte buffers. In this formulation, water is added as an acidic Immobiline having pK = 14 and a unit molar concentration (or with a pK = 15.74 and standard 55.56 molarity) since around pH 11 its buffering power becomes significant. The gel contains a 'conductivity quencher', i.e. a density gradient incorporated in the matrix, with the dense region located on the cathodic side (pH 11) for (a) smoothing the voltage gradient on the separation cell and (b) reducing the anodic electrosmotic flow due to the net positive charge acquired by the matrix at pH 11 (1 mM excess protonated amino groups to act as counterions to the 1 mm OH- groups in the bulk water solution generated by the local value of pH 11). Excellent focusing is obtained for such alkaline proteins as lysozyme (pI 10.55), So-6 (a leaf protein, pI 10.49), cytochrome c (pI 10.45) and ribonuclease (pI 10.12).     

Preparative isoelectric focusing in immobilized pH gradients. I. General principles and methodology

A new method for preparative protein purification is described, based on the use of Immobiline matrices. After electrofocusing, the protein zone of interest is recovered by electrophoretic transfer to a hydroxyapatite gel, from which it is eluted with 0.2 M phosphate buffer, pH 6.8, with yields for the proteins studied in the range 76-98%. For six different proteins, the focusing step gives a common upper limit of approximately 45 mg protein/ml gel as mean concentration in a focused protein zone. It is demonstrated that in practical preparative work, components with a pI difference of 0.007 pH units can be completely resolved, and that on a 5-mm-thick gel of dimensions 240 X 110 mm, samples containing as much as 400 mg of the major protein component can be applied. Focusing of large amounts of a salt-containing sample is demonstrated with the aid of human serum. A theoretical expression is given relating the concentration distribution and maximum protein concentration within a focused zone to the applied voltage, the pH slope used and the zone width. Based on this expression and the finding of an upper concentration limit for a protein we shown how to optimize the parameters in preparative work with immobilized pH gradients in relation to the separation power needed. Finally, it is shown that, in comparison with conventional preparative electrofocusing in polyacrylamide gels, immobilized pH gradients allow a ten-fold increase in load, whilst still giving a resolution comparable to that of analytical isoelectric focusing.     

Molecular forms of guanine aminohydrolase (author's transl)

Guanine aminohydrolase (E.C. 3.5.4.3) has been purified 11-fold from the supernatant fraction of guinea-pig liver homogenates in 0.25 M sucrose (centrifuged at 50,000 X g) through thermic denaturation at 60 degrees C and ammonium sulphate fractionation (30--60% saturation). The enzyme in the homogenates and purified preparations exhibited two Km values. In both preparations four enzymatic electrophoretic bands have been detected. Purified guanine aminohydrolase is chromatographically resolved on DEAE-sephadex in three components whose active forms appeared separately on their pherograms. The enzymatic form eluted at lower ionic strength has the least anodic mobility, is inhibited by guanine (4 X 10(-5) M) and presents only one Km value (1.5 X 10(-5) M). The enzymatic form eluted at greater ionic strength exhibits the highest anodic mobility, is also inhibited by guanine (7 X 10(-5) M) and its Km value seems to be 6.3 X 10(-6) M. Molecular weight of enzymatics forms determined by Sephadex G-200 chromatography, is 120,000 +/- 5,000. The preceding results, correlated with the chromatographic homogeneity of guanine aminohydrolase, purified in Sephadex G-100, suggests that the four molecular forms of the native enzyme may be considered as isozymes.     

Preparation of polypeptide subunits of cytochrome oxidase from Neurospora crassa.

Cytochrome oxidase was purified from Neurospora crassa by ammonium sulfate fractionation in the presence of bile salts. The enzyme preparations contained 10-13 nmol of heme a per mg of protein; no other hemoproteins could be detected. Dodecylsulfate gel electrophoresis resolved the enzyme complex into seven major bands, representing seven polypeptide subunits. A procedure is described that allows the isolation of these enzyme subunits on a large scale starting from a single batch of oxidase preparation. It involves dissociation of the enzyme complex by dodecylsulfate and subsequent separation of the obtained polypeptides by chromatography in the presence of various dodecylsulfate concentrations. Purification of subunits 3, 4, 5, 6 and 7 was achieved by column chromatography using molecular sieves (Sephadex G-100, Bio Gel P-60) and hydroxylapatite. For the purification of subunits 1 and 2 an electrophoretic separation on a preparative polyacrylamide gel was required. The advantages and disadvantages of the separation procedure of the enzyme polypeptides are discussed. As a special point of interest, the conservation of antigenic determinants of the polypeptide chains during the dodecylsulfate treatment is considered.     

Characterization of polymeric buffers for operating membrane-trapped enzyme reactors in an electric field

A novel class of amphoteric, polymeric buffers, is described, consisting of grafting onto growing polyacrylamide chains weakly acidic and basic acrylamido-monomers (called Immobilines; protolytic groups as N-substituents on the nitrogen of the amido bond), for operating a membrane-immobilized enzyme reactor (MIER) in an electric field. With these soluble, polymeric buffers, it is possible to operate the membrane reactor at any optimum of pH activity, for any given enzyme, in the pH 3-10 scale. Such buffers, being amphoteric, are confined in the enzyme reaction chamber by the same isoelectric trapping mechanism. The best buffers were found to be those polymerized in presence of 9% neutral monomer (acrylamide) and containing 20 mM Immobiline as buffering ion. To decrease their viscosity in solution, the polymeric buffers are synthesized at high temperatures (70 degrees C) and in presence of a chain-transfer agent. The weight average molecular size in these conditions has been found to be ca. 200,000 Da. These buffers exhibited excellent performance in a variety of enzyme reactions in the MIER, such as in the case of penicillin G acylase and histidine decarboxylase and were found to greatly stabilize enzyme activity, permitting operation of the MIER over extended periods of time. As an example, in a penicillin G acylase reactor, >75% enzyme activity was maintained over a 10-d cycle of operation, while with conventional buffers more than 90% inactivation was experienced over the same period of time. This novel class of macromolecular, amphoteric buffers could also be exploited in other types of conventional bioreactors not based on an isoelectric trapping mechanism.     

Enzyme reactions in a multicompartment electrolyzer with isoelectrically trapped enzymes

The possibility of performing bioconversions under an electric field is here reported. A system is described by which the enzyme is trapped by an isoelectric mechanism between two zwitterionic membranes having pI values encompassing the isoelectric point of the enzyme. The enzyme is loaded into a multicompartment electrolyzer and kept operating under an electric field, which will continuously harvest the reaction product. Since, under focusing conditions, all buffering ions will vacate the reaction chamber at steady state, the buffering ion is trapped into the enzyme chamber by using amphoteric buffers co-isoelectric with the enzyme. As an example of such ‘isoelectrically immobilized’ reactor, the enzyme β-hydroxysteroid dehydrogenase is blocked into an isoelectric trap delimited by a pI 8.0 and a pI 6.5 membranes. 100 mM histidine (pI 7.47) is co-immobilized by the same isoelectric mechanism into the enzyme chamber. The dehydrocholic acid substrate (3,7,12-trioxo-5β-cholanoic acid) and reduced co-factor (NADH) are continuously infused into the enzyme chamber and the product (3β-hydroxy-7,12-dioxo-5β-cholanoic acid, a compound of pharmaceutical interest) and the oxidized co-factor (NAD⁺) collected, separately, into the two neighbouring chambers at the anodic side. Advantages: in a soluble form, the enzyme maintains the reaction kinetics of the free soluble form. Additionally, the reaction product and exhausted co-factor can be recovered by electrophoretic transport.     

PURIFICATION OF BOVINE PINEAL HYDROXYINDOLE O-methylTRANSFERASE BY IMMUNOADSORPTION CHROMATOGRAPHY

A procedure is described for the use of immunoadsorption chromatography of hydroxyindole O-methyltransferase (HIOMT). HIOMT was purified from bovine pineal extract by affinity chromatography on immunoglobulins (Ig)-Sepharose. The overall purification was about 45-fold; the yield was 84%. This enzyme constitutes about 2.0% of the soluble proteins in the pineal gland. The enzyme represented a single precipitin line on Ouchterlony double diffusion plate and immunoelectrophoresis. Ultracentrifugation analysis indicated the existence of molecular aggregates of enzyme and disc gel electrophoresis showed one main protein band and several minor bands. However sodium dodecyl sulphate (SDS) gel electrophoresis showed a single protein band with subunit molecular weight 38,000 demonstrating bovine pineal HIOMT to be polymer enzyme of a single subunit. The properties of the purified enzyme including disc gel electrophoretic pattern, the effect of pH, chemicals and substrates and immunological properties were identical with those of the crude enzyme.     

Preparative isoelectric focusing in immobilized pH gradients. II. A case report

The preparative aspects of isoelectric focusing (IEF) in immobilized pH gradients (IPG) have been investigated as a function of the following parameters: environmental ionic strength (I), gel geometry and shape of pH gradient. As model proteins, hemoglobin (Hb) A and a minor, glycosylated component (HbA1c), with a delta pI = 0.04 pH units, have been selected. The load capacity increases almost linearly, as a function of progressively higher I values, from 0.5 X up to 2 X molarity of buffering Immobiline (pK 7.0) to abruptly reach a plateau at 3 X concentration of buffering ion. The load capacity also increases almost linearly as a function of gel thickness from 1 to 5 mm, without apparently levelling off. When decreasing the pH interval from 1 pH unit (pH 6.8-7.8) to 1/2 pH unit (pH 7.05-7.55) the amount of protein loaded in the HbA zone could be increased by 40%. In 5 mm thick gels, at 2 X pK 7.0 Immobiline concentration, over a 1/2 pH unit span, up to 350 mg HbA (in a 12.5 X 11 cm gel) could be loaded in a single zone, the load limit of the system being around 45 mg protein/ml gel volume.     

Purification and characterization of a beta-glucosidase from Trichoderma reesei

A beta-glucosidase has been purified from culture filtrates of the fungus Trichoderma reesei QM9414 grown on microcrystalline cellulose. The beta-glucosidase was purified using two successive DEAE-Sephadex anion-exchange chromatography steps, followed by SP-Sephadex cation-exchange chromatography and concanavalin-A--agarose chromatography. Evidence for homogeneity is provided by polyacrylamide disc gel electrophoretic patterns, which show a single protein band. Sedimentation equilibrium analysis yielded a molecular mass of 74.6 +/- 2.4 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a single protein band with a molecular mass of 81.6 kDa. Thus, the enzyme appears to be a single, monomeric polypeptide. The beta-glucosidase is isoelectric at pH 8.5. The enzyme is rich in basic amino acids and contains few half-cystine and methionine residues. The purified beta-glucosidase contains less than 1% by weight of neutral carbohydrate. The beta-glucosidase catalyzes the hydrolysis of cellobiose, p-nitrophenyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-glucopyranoside; the values of V/Km for each substrate were determined to be 2.3 X 10(4), 6.9 X 10(5) and 2.9 X 10(6) M-1 S-1 respectively. The enzyme is optimally active from pH 4.5 to 5.0 and is labile at higher hydrogen ion concentrations. The beta-glucosidase has an unusually high affinity for D-glucose (Ki = 700 microM). Comparison of inhibition constants for cello-oligosaccharides suggests that the substrate-binding region of the beta-glucosidase comprises multiple subsites.     

Oxytocinase-like enzyme in an ovarian dysgerminoma: a placenta-specific protein

Aminopeptidase from dysgerminoma was purified and characterized using L-leucine-beta-naphthylamide as substrate. The enzyme was resistant to puromycin, methionine, amastatin, bastatin, and EDTA, and it was heat labile at 60 degrees C. The enzyme showed the same electrophoretic mobility as pregnant-patient serum oxytocinase CAP1 on polyacrylamide gel electrophoresis. Km value against S-benzylcysteine-p-nitroanilide was 4.2 X 10(-4) M. Oxytocin and vasopressin competitively inhibited the enzyme activity. Molecular weight of the enzyme was estimated to be 80,000 by Sephadex G-200 column chromatography. These results suggest that aminopeptidase from dysgerminoma is an oxytocinase-like enzyme, a placenta-specific protein.     

Physicochemical properties of a beta-lactamase from Streptomyces UCSM-104

A purified beta-lactamase from Streptomyces UCSM-104 shows the presence of three subforms when stained for protein and/or for activity after polyacrylamide gel electrophoresis or after electrofocusing. The pI values of the three subforms were 5.45, 5.30 and 5.10, respectively. The respective electrophoretic mobilities were 4.6 X 10(-5), 5.2 X 10(-5) and 5.9 X 10(-5)m2/sV. Relative molecular mass of 14,900 was determined. The amino acid composition was established. Cysteine was not detected. A fairly high proline content (8.3%) differentiates this enzyme from other beta-lactamases. Lysine was the only N-terminal amino acid detected after dansylation. The possible origin of the subforms is discussed.     

Isolation of the zona pellucida and purification of its glycoprotein families from pig oocytes

The isolation of the porcine zona pellucida, the glycoprotein envelope surrounding the mammalian oocyte, and the purification of its glycoprotein families is described. The zona pellucida was prepared from oocytes isolated from pig ovaries using a razor blade device and sieving procedures with Teflon or nylon screens. In 6-7 man h, the zona pellucida from 5 X 10(5) oocytes was obtained yielding 12 mg protein and 2.2 mg carbohydrate. The absorptivity of heat solubilized and filtered zona pellucida was A1%280 nm, 1 cm = 10.8. The four glycoprotein families of the zona pellucida were purified by two-dimensional polyacrylamide gel electrophoresis and electrophoretic elution. The electrophoretic purity of these families was greater than 90%. The protein and carbohydrate content and the amino acid and monosaccharide compositions of each of the glycoprotein families were determined.     

Isoelectric protein purification in segmented immobilized pH gradients. Effect of salts on rate of contaminants' removal

A method is described for keeping a constant salt background during protein purification in a segmented immobilized pH gradient. It is based on an external hydraulic flow replenishing the salt loss due to combined electric and diffusional mass transport (similar to the concept of Ribes' steady-state rheoelectrolysis). Such a minimum of ionic strength might be needed for proteins which tend to precipitate and aggregate at or in vicinity of the isoelectric point. However, it is found that any salt level in the sample feed (already at 1 mM concentration) deteriorates transport of non-isoelectric proteins, because of the much larger current fraction carried by the ions themselves as opposed to proteins. In addition, high salt levels in the sample reservoir might form cathodic and anodic ion boundaries, alkaline and acidic, respectively, which might hamper protein migration and even induce denaturation. Thus, when high salt backgrounds are needed in the sample feed, external pH control should be exerted, e.g. with a pH-stat. Three parameters influence protein transport in the segmented IPG chamber: (a) cross-sectional area of the Immobiline membranes; (b) delta pI between the isoelectric protein and the contaminants and (c) salt molarity in the sample reservoir. The first 2 show a positive, the last a negative correlation.     

Rapid electrotransfer of proteins from polyacrylamide gel to nitrocellulose membrane using surface-conductive glass as anode

A new type of inexpensive horizontal apparatus for the electrophoretic transfer of proteins from a gel to an immobilization membrane has been developed. In this system, gel and membrane were directly pressed between two flat electrodes. A surface-conductive glass was used as anode and a stainless-steel plate as cathode. Proteins could be transferred from polyacrylamide gel to nitrocellulose sheet, with a yield of at least 90% in 60-90 min, without overheating, using a voltage gradient of 30-40 V/cm. Moderate volumes of separate anodic (with 20% methanol) and cathodic (with 0.1% sodium dodecyl sulfate) buffers were suited for optimal transfer of proteins with relative molecular mass (Mr) in the 14,000-94,000 range.     

Purification and characterization of heparinase from Flavobacterium heparinum

Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.     

Esterases of developing human brain

Abstract The free and bound nonspecific esterases occurring in, respectively, the saline and triton X-100 extracts of adult and developing human brain were studied by starch gel electrophoresis (zymograms). Zymograms of the free esterase fraction visualized with NA as substrate were qualitatively similar at 5-12 days of age to the electrophoretic patterns observed in adult material. In both adult and developing brain, zymograms of bound esterase resembled those of the free enzyme, the major difference being the presence in the former of a slow, broad, anodic zone of diethyl-p-nitrophenyl phosphate-inhibited enzyme. Esterases characteristic of adult white matter and having preferential affinity for alpha-naphthylpropionate, alpha-naphthyl butyrate and alpha-naphthyl valerate were not identified in infant brain until about 4 months postnatal age. A far-moving, anodic enzyme was distinctly evident in zymograms of brains of less than 1 month of age. This enzyme hydrolysed NP, NB, and NV more actively than alpha-naphthyl acetate. It was present in the adult brain but, in contrast to the infant, was no longer electrophoretically-separable from another enzyme which had greater affinity for NA and had previously been designated the A₁₀ band. Quantitative assays demonstrated that the bound esterase increased in cerebral and cerebellar cortex during development. In contrast, the proportion of free to bound esterase showed little change in white matter. Acetylcholinesterase and butyrylcholinesterase zymograms became identical to adult patterns from 1 to 4 months of age. Thiolacetic acid esterase was present at 38 weeks gestation. Some functional and anatomical correlations were attempted in explanation of the biochemical observations.