Isoelectric Zone Research Articles

In situ photo-immobilised pH gradient isoelectric focusing and zone electrophoresis integrated two-dimensional microfluidic chip electrophoresis for protein separation

A method is introduced for open-column photo-induced site-selective immobilization of pH gradients in a layer of PEG-methacrylate in a multi-dimensional microfluidic chip for use in electrophoresis. It has several attractive features: (a) mixtures of fluorescently labelled proteins carbonic anhydrase, catalase and myoglobin in their native state can be separated by pH-gradient isoelectric focusing (IEF) and zone electrophoresis (CZE) using integrated 2D chip electrophoresis; (b) compared to strip packing or monolithic photo-immobilization, it overcomes the shortcomings of free carrier ampholyte-based 2D chip electrophoresis in an easy way; (c) larger amount of sample can be loaded into the open column-mode electrophoresis (d) immobilized pH gradients can be re-used and the chip can be recycled; (e) a multilayer 3D pH gradient is established by a layer-by-layer assembly technique to further increase the separation capacity. In our perception, this strategy has a large potential in microfluidic chip-based separation schemes because of its simplicity, separation power, re-usability, and separation capacity.

Recent developments in capillary and chip electrophoresis of bioparticles: Viruses, organelles, and cells

In appropriate aqueous buffer solutions, biological particles usually exhibit a particular electric surface charge due to exposed charged or chargeable functional groups (amino acid residues, acidic carbohydrate moieties, etc.). Consequently, these bioparticles can migrate in solution under the influence of an electric field allowing separation according to their electrophoretic mobilities or their pI values. Based on these properties, electromigration methods are of eminent interest for the characterization, separation, and detection of such particles. The present review discusses the research papers published between 2008 and 2010 dealing with isoelectric focusing and zone electrophoresis of viruses, organelles and microorganisms (bacteria and yeast cells) in the capillary and the chip format.

Solid–liquid separation by sonochemistry: A new approach for the separation of mineral suspensions

The effect of sonochemistry to acidify solutions was applied for the solid–liquid separation of three kinds of mineral suspensions. At first, the relationship was measured between zeta-potential and pH in these suspensions to find pH levels correspondent to the isoelectric points. Then sonication (200 kHz or 28 kHz) was applied to adjust pH to the isoelectric points and separated particles from solutions by still-standing and spontaneous precipitation. Compared to the conventional methods using filters and chemical agents, the advantage of this sonochemical separation is two-fold. First, it does not require the maintenance of filters. Second, separated particles are easy to use since they are not mixed with pH adjusters and chemical flocculants. Isoelectric zone (ion strength 0.01, concentration 0.001 wt.%) of green tuff, andesite and titanium dioxide suspensions tested in this study were pH 1.1–3.7, 0.8–3.4, 2.7–5.7, respectively. The sonication of green tuff and andesite suspensions at 200 kHz changed the pH to the isoelectric zone despite the pH buffering effect of eluted alkali earth metals, and successfully precipitated the particles. On the contrary, the sonication of these suspensions at 28 kHz failed to adjust pH to the isoelectric zone, and the particles did not precipitate. In addition, the degradation of particles was observed in the SEM photographs of particles sonicated at 28 kHz, whereas no significant change was detected in particles sonicated at 200 kHz. Thus, it is concluded that the optimal frequency is about 200 kHz because its strong chemical effect can easily adjust the pH while its relatively weak physical effect prevents the degradation of particles.

Monoclonal immunoglobulins: affinity blotting for low concentrations in serum.

I describe a simple, economical technique for identifying low concentrations of monoclonal immunoglobulins in the presence of excessive amounts of immunoglobulins of other classes. The technique involves binding of specific antibody to nitrocellulose, separating proteins by isoelectric focusing or zone electrophoresis in agarose gels, using capillary transfer to bind proteins to the nitrocellulose via their antibody affinity, and then detecting transferred proteins with enzyme-labeled antibody. A monoclonal immunoglobulin can be completely characterized in 2 h. No expensive equipment is required. Affinity blotting is about 10-fold as sensitive as native blotting, 100-fold as sensitive as silver staining.

Emulsifying Properties of Bovine Blood Globin: A Comparison with Some Proteins and Their Improvement

The emulsifying properties of bovine blood globin were compared with those of bovine serum albumin, hemoglobin and ovalbumin. Although the emulsifying activity of globin was greater than that of hemoglobin and ovalbumin, it decreased in the isoelectric zone owing to the low solubility of globin. Acetylation increased the emulsifying activity of globin in the isoelectric zone, but decreased in the acidic pH region. Pepsin digestion did not increase the emulsifying activity of globin, but addition of CMC to pepsin-digested globin improved both its emulsifying activity and the emulsion stability.

Effects of Ca++, Mg++ and Na+ on Heat Aggregation of Whey Protein Concentrates

ABSTRACTEffect of Ca++ on the heat aggregation of whey protein concentrates (WPC) was compared with that of Na+ and Mg++. On the alkaline side of the isoelectric zone, aggregation of WPC was increased by the addition of CaCl2, MgCl2 or NaCl, among which CaCl2 showed the greatest effect. The denaturation temperature of WPC determined by differential scanning calorimetry significantly decreased in the presence of CaCl2 or MgCl2, but increased slightly in the presence of NaCl. In the electrophoretic patterns of heated WPC, the most sensitive protein to Ca++ was β‐lactoglobulin.

Molecular size, subunit structure and microheterogeneity of glycoprotein II from the seeds of kidney bean ( Phaseolus vulgaris L.)

The isolation of Glycoprotein II from the seeds of kidney bean ( Phaseolus vulgaris) cv. ‘Processor’ is described. This glycoprotein was shown by SDS-gel electrophoresis to dissociate into four subunits, 53 000, 50 000, 47 000 and 43000 (α : β : μ : δ), in an approximate ratio of 2 : 0.2 : 2 : 1. At neutral and slightly alkaline pH values its molecular weight was about 142 000 (protomer) while at pH 5 it was mainly in the form of a tetramer with a molecular weight value of about 560 000. Samples of Glycoprotein II were shown by isoelectric focusing, molecular sieve chromatography and immunochemical methods to be microheterogenous. A number of fractions were prepared by these methods in which the proportion of the major subunits (α : γ : δ) varied between 1 : 1 : 1 to 3 : 3 : 1. These ratios were also shown to change during development of the seed. On the basis of these results it is suggested that, at and above pH 7, samples of Glycoprotein II consist of a microheterogenous population of molecules each containing three subunits per protomer. However, in these protomers the subunits are drawn, in different ratios, from the four subunits available. When the net molecular charge is small, at and around the isoelectric zone of Glycoptotein II, the protomer is converted mainly into the more stable tetramer containing 12 subunits.

Preparation and Properties of 2-Amino Acid Type Amphoteric Surfactant Containing Hydroxyl Group. I

2- [N- (2-hydroxyethyl) amino] fatty acid and 2- [N, N-bis (2-hydroxyethyl) amino] fatty acid were prepared by a reaction of 2-bromo methyl ester of fatty acid with monoethanolamine or diethanolamine. Where fatty acids employed were decanoic lauric, myristic, palmitic, and stearic acids. The cmc, the pH-dependence of surface tension and the isoelectric point were measured. The cmc of 2- [N- (2-hydroxyethyl) amino] fatty acid was lower in the order of C10>C12>C14. On the other hand, one of 2- [N, N-bis (2-hydroxyethyl) amino] fatty acid was in the order of C10>C12>C14>C16>C18 and was presented by the formula of log cmc=0.52-0.266 N. Where N was a total carbon number. From the result of surface tension measurement against different pH, 2- [N, N-bis (2-hydroxyethyl) amino] fatty acids with C14 and C16 alkyl chain length had the most steady surface activities in all pH. All these amphoteric surfactants were found to have a wide expanded isoelectric zone and these surface ranges. tensions did not lower even in the isoelectric point probably owing to the effect of the hydroxyl group.

Solubility and Heat Stability of Whey Protein Concentrates

Rennet whey protein concentrates have excellent nutritional properties, but their use in fluid food systems is impaired by the poor heat stability of the protein. Heating whey protein concentrated solutions at neutral pH caused up to 70% loses in solubility. In the absence of added calcium, protein coagulation occurred near the iso-electric zone whereas in the presence of .03M calcium chloride, similar protein coagulation occurred in the whole pH range (pH 2 to pH 12). Tryptic hydrolysis of the protein increased the heat stability of whey protein concentrates considerably.

Characterisation of Escherichia coli cell surface by isoelectric equilibrium analysis

A characterisation of the lipopolysaccharide (outermost) layer of Escherichia coli cells has been made by isoelectric equilibrium analysis. Unmodified E. coli cells show a surface isoelectric point (pI) of 5.6. Cells treated with ethyleneimine in order to esterify the carboxyl groups are isoelectric at pH 8.55. When amino groups are blocked the bacterial surface has a pI of 3.85. An analysis of these results suggests that the ionisable groups occurring in the isoelectric zone i.e. the zone amenable to investigation by the isoelectric equilibrium method are: carboxyl groups and amino groups of polysaccharide and protein components. The carboxyl groups have a p K between 3.2 and 4.5 and the amino groups have a p K of 7.5. ε-Amino groups, phenolic hydroxyl groups and guanidyl groups do not occur, and phosphate and amino groups of the phospholipid complex are not detected. The number of thiol groups in the isoelectric zone has been determined using 6,6′-dithiodinicotinic acid. The number of anionogenic and cationogenic groups has been determined. From the density of the negative charges on the surface it is estimated that the isoelectric zone might extend up to 60 Å below the cell surface. The data discussed in this paper relate to the outermost layer of the bacterial cell wall composed of lipopolysaccharide-phospholipid-protein complex. Since reactive groups of the phosphilipid component of the complex have not been detected in the isoelectric zone, it is suggested that the arrangement of lipopolysaccharide phospholipid protein complex is such that the phospholipids are located at a depth of more than 60 Å from the bacterial surface.

ミオシン区蛋白質ゲルの保水性-I

Water holding capacity (WHC) of myosin-fraction (“M”) gel was determined under varied pH conditions by centrifugal method (8, 000rpm, 20 min.). 1. WHC of “M” gel is greater than that of muscle homogenate, being maximum in isoelectric zone in contrast with the latter which is minimum in this region. 2. WHC of isoelectric “M” gel decreases if pH is shifted to ?? 4.5 or ?? 8. Similarly decreases WHC of “M” gel formed in the alkaline side on readjustment of pH to ?? 4.5. 3. If pH of “M” gel formed in acid or alkaline side is brought to isoelectric zone, WHC is not only less than that of isoelectric gel, but also of the original gel. 4. If pH of isoelectric or alkaline gel is brought to ?? 4.5, WHC of the reformed gel is smaller than that of gel formed at pH ?? 4.5. Results 1 and 2 might be interpreted by an assumption that firmness of structure sufficient to hold water against pressure applied in measurement of WHC has a decisive influence on WHC in the case of gels with relatively weak structure as “M” gel. In Result 3 WHC decreases, though the structure of reformed gel is supposed to be thickened by pH adjustment. This might be probably due to that syneresis has more pronounced effect than firmness of structure. Result 4 can not be explained either by loosening of structure nor syneresis.

Interaction between proteins and salt solutions. III. Effect of salt type and concentration on the shrinkage temperature

AbstractThe effect of salt type and concentration on the transformation of an oriented crystalline collagen tendon into a crosslinked network under conditions of equilibrium swelling was investigated. Our main observations are the following. The degree of swelling of crystalline tendons increases at low salt concentration Cs, and decreases at higher Cs for a wide variety of salts. The observation is not reconcilable with swelling taking place in interfibrillar spaces or structural voids. Within the tropocollagen units and at their ends, regions of reduced organization are postulated (as suggested by Bear and by Schmitt) which are able to interact with the diluent in the amorphous‐like manner. At least four different factors should be considered in assessing the role of salt and salt concentration on the shrinkage temperature Ts under isoelectric conditions. They are: (1) specific effects, (2) diluent effects, (3) crosslinking effects, and (4) nonequilibrium effects. The diluent effects are correlated with the salting‐in–salting‐out power of the ions which was characterized in Part I of this series. Smaller amounts of diluents are generally available to the tendon when the salt has a higher salting‐out power, and this corresponds to higher shrinkage temperatures, other conditions being the same. The crosslinking effect raises Ts due to a reduction of the diluent content and, probably for p‐benzoquinone and formaldehyde, also to a reduction of the conformational entropy in the molten state. Nonequilibrium effects arise from the fact that shrinkage and recrystallization are kinetically hindered when the tendon is highly deswollen in strong salting‐out solutions, or when the salt has a crosslinking power. The specific effect is the only effect which is not related to the amount of diluent present in the tendon. Its origin is less clear. For anions such as Cl− and SCN−, it is possibly related to an ability of the ion to prevent intersegmental hydrogen bonding and water carbonyl bridges. The competition of several of the above effects for a given salt solution makes possible various types of dependence of Ts upon Cs: Ts may either continuously decrease or continuously increase with increasing Cs, or it may go through a minimum. In absence of salt, the cooperative character of the transition at the pH at which maximum swelling occurs appears extremely reduced. The large swelling maintains the tendon in the elongated state and this simulates a continuous decrease of Ts on lowering pH. In presence of small quantities of salt, which reduce swelling, the transition is sharp and Ts is decreased with pH up to pH 2, when maximum swelling occurs, and then reincreases on further lowering of the pH. The dependence of Ts upon Cs is more complex than under isoelectric conditions. There is generally an increase of Ts with Cs which is equivalent to an increase of the denaturation temperature with Cs for helical polyelectrolytes in solution. At higher salt concentrations, however, Ts may decrease again, and possibly increase again at still higher salt concentrations, depending upon the effect of the salt solution in the isoelectric zone.

Some Properties of the Fibrillar Proteins of Normal and Watery Pork Muscle

SUMMARYIt is shown that the washed muscle fibrils obtained from watery pork have a lower water retention at low ionic strength, and much lower extract‐ability at high ionic strength, than the fibrils from normal pork. These changes are accompanied by a gain of protein by the washed watery fibrils, and this protein originates from the soluble sarcoplasmic proteins. All the changes, including the characteristic gain of protein, can be artificially induced in normal meat by allowing it to pass into rigor at 37° C. The isoelectric region, or region of minimum swelling of watery fibrils, whether washed or unwashed, is similar to or slightly lower than that of normal fibrils. There is a broad isoelectric zone in both cases, extending from ∼pH 5 to ∼5.70. On the other hand, the IP of fully coagulated fibrils lies between 5.6 and 6.1. Washed and unwashed fibrils of watery meat show about the same degree of swelling at all pH values. Normal fibrils, however, show a higher water retention in the unwashed state than the washed. This effect is not due to the Mg or Ca ions included in the unwashed samples, but may result from interaction between the sarcoplasmic and fibrillar proteins. In the unwashed state, the swelling of normal fibrils is nearly double that of the watery fibrils at all pH values.It is shown that the rise of pH in intact carcasses of watery meat as they cooled from 37 to 10° C was probably due to the effect of temperature on the pK of ionizable groups of the proteins and buffering substances. It can be reproduced artificially and reversibly in native and coagulated minced meat, merely by raising or lowering the temperature. The titration curves of watery fibrils show similar titration constants (pK') to those of normal fibrils, but a loss of ratable groups. Heat coagulation, on the other hand, results not only in a bigger loss of titratable groups but in a much larger shift in the titration constants. These results can be interpreted to show that the fibrillar proteins of the watery fibrils are not denatured or aggregated in the usual sense, but are probably covered by a layer of denatured sarcoplasmic protein that is firmly‐bound to the surface of the myofilaments.

Microeletrophoretic studies of soluble collagen

The electrophoretic mobility of soluble collagen of rat tail tendon, adsorbed on oil droplets, has been measured in a range of salt solutions between pH 1 and pH 12, maintained at constant ionic strength. Specific effects of the lower valent buffer ions used appear small and the corresponding mobilities lie on a smooth curve. These mobilities parallel the hydrogen ion binding expected from the amino acid composition, with a long isoelectric zone in the range pH 7.7 ± 0.8, in agreement with reported isoelectric points of native collagen. Addition of the divalent cations: Pb ++, Cu ++, Ca ++, Mg ++, or ofthe anions: citrate, phosphate, pyrophosphate, oxalate, at constant ionic strength shifts the mobility, in order of decreasing effect, to more positive or negative values respectively, with consequent shortening and raising or lowering of the isoelectric zone. These specific effects are attributed to binding of these ions by the protein, and the quantitative interpretation is discussed.

ゼラチン

As often reported, if limited quantities of water and gelatin xerogel are brought into contact with each other there takes place a contraction in total volume. As to the dependence of this shrinkage on the pH value of the liquid that surrounds the gel, however, a group of investigators have found that the volume decrease attains to a maximum in a pH region about the isoelectric point of the gelatin, while another group say that it becomes minimal there; but any convincing theory seems to have not yet been put forward to resolve this antinomy. The present author, intending to find a clue to the molecular events inducing the spontaneous wetting of extremely fresh fillet, examined as preliminaries the said contraction of a gelatin-water system and found that the contraction minimum in the isoelectric zone is just realized only at a lower temperature. He proposes, hereupon, a view that the apparent volume reduction may be caused by the resultant of two separate mechanisms, viz., (1) fixing of water molecules around rather stationary polar groups of gelatin, which reduces the space thermally swept by these molecules, and (2) migration of water molecules into oil-repellent gaps in the xerogel, which lessens the water remaining outside the gel. Results of the present experiment, in which the contraction dependence on pH of the immersion liquid was examined at different temperatures and with varying moisture-contents of sample, will serve by the help of this view to settle the pending antinomy about the contraction of a gelatin-water system.

魚介肉の膨潤-I

In order to obtain in a simple method knowledge of hydration of fish muscle which plays an important role in behavior of the muscle proteins, a series of research has to be carried out on swelling of muscle. In the present study, effect of pH on swelling of comminuted muscle is examined. Degree of swelling (S) is expressed by volume percentage of sedimentary muscle in a mixture of I part muscle and 3 parts water after centrifuged. In case of horse mackerel muscle, on either side of an isoelectric zone (about 5.0-6.0), addition of HCl up to about pH 4.5, or of NaOH up to about pH 9.5 occasions very pronounced swelling. Further addition of acid or alkali causes slight peptization and the swelled muscle shows weak fluidity (Fig. 1). KCl, added so as to 0.5 M solution, suppresses swelling of muscle caused by HCl below an apparent isoelectric point, pH 5.5 (pH for the minimum swelling), but above pH 5.5, it exerts an exactly opposite effect, namely, very marked increase in swelling is observedin presence of salt (Fig.4). From these facts it is deduced that swelling of muscle cannot be explained simply on basis of osmotic pressure alone, but it seems to have a close relation with hydration of muscle proteins, especially that of myosin. Because solubility of myosin'rises on the alkaline side, while decreases on acid side of isoelectric point in presence of salt. (7) According to the nature of anions or cations, different kinds of acids or bases vary in their effects upon the swelling. For instance, H2SO4 (Fig. 2) and Ba (OH)2 (Fig. 3) which both interact with the proteins as a bivalent ion, do not cause so marked swelling as HCl or NaOH. When compared the swelling of horse mackerel muscle with those of sardine, carp, and skip-jack, no appreciable differences are observed among the behaviors of their swelling. On the other hand, the muscle of squid behaves differently from the other fish muscles in the following ways: (1) its isoelectric point (pH 4.8) is lower than that of the others (pH 5.5), (2) marked swelling is observed between 5.5 and 8.5, (3) when pH stands outside of this renge, considerable peptization of the proteins occurs to form a viscous solution (Fig. 5).

The Solar–Activated Flavor of Homogenized Milk. IV. Isolation and Characterization of a Whey Constituent Capable of Producing the Solar–Activated Flavor

Summary A minor-protein fraction has been isolated from skimmilk after the major proteins had been removed, which is capable of being photosensitized to produce the typical solar-activated flavor of homogenized milk. The elementary analysis, ammo acid composition, isoelectric zone and the minimum molecular weight ( M min.) have been determined. From the data, a ( M min.) of 70,300 was calculated along lines of orthodox organic chemistry. The determination of 12 amino acids shows that the minor-protein fraction contains the following amino acid residues: Arg 7 , CyS 5 , Glu 64 , His 4 , Ileu 20 , Leu 31 , Lys 28 , Met 6 , Phe 10 , Thr 39 , Try 3 and Val 59 (the first 3 letters of each amino acid is used as the symbol).

Studies in Wool Dyeing: Comparison of Metheds for Applying Chrome Dyes

When a wool staple is dyed with certain chrome dyes the tip portions are heavily dyed and the body portions are undyed or only lightly dyed. Cross‐sectional examination shows that the tip portions of such a staple are heavily dyed, the body portions are undyed and the intermediate portions are “ring” dyed, the thickness of the “rings” being greater toward the tip portions. This variation in dye penetration is caused by the condition of the epidermis of the wool fibre, and if this epidermal layer is broken or “damaged” the dye will penetrate the fibre more readily. When the dyed staple is carded or when wool is dyed after carding, the cross‐sections show unlevel distribution of dye from fibre to fibre. This fibre unlevelness causes poor colour values, skittery or heathery effects, and entails an inefficient use of dye. Exhaustion curves show that there are variations in wools from different regions and after physical and chemical processing. These variations increase the possibility of fibre unlevelness when various wools are blended together.Wool exhibits reactivity in aqueous solutions and induo.09 changes in pH both above and below the isoelectric zone, viz. pH 4–5. When a wool dyebath is made alkaline with ammonia at the start of the dyeing operation there is a gradual change in pH during the dyeing process, which influences the chromation, the decomposition of dye by oxidation and the exhaustion.Wool can be successfully dyed with chromable dyes which are not usually applied by the metachrome methed. This can be accomplished by dyeing in a bath which initially has a pH greater than 7, by using an optimum low percentage of bichromate, by having magnesium sulphate present, and by employing a surfaceactive agent as a dyeing assistant. This procedure has been called the Calcomet process. The addition of magnesium sulphate to the alkaline dyebath results in the formation of a magnesium‐dye complex without appreciably affecting the pH. The magnesium complex, however, appears to be unstable below a pH of about 7. The use of the specially designed surface‐active agent as a dyeing assistant promotes dye exhaustion from the alkaline bath and also causes the exhaustion of any chromium‐dye complex that is formed in the bath. Consequently, good penetration and levelness of shade are obtained.The afterchrome, chrome mordant, metachrome and Calcomet metheds were compared in the laboratory using several chrome dyes, and a detailed comparison of the various metheds was made with Calcochrome lied ECB in regard to rate of dyeing, rate and extent of chromation, pH and the completeness of exhaustion.

Effect of pH and NaCl on Swelling and Drip in Fish Muscle

Sodium chloride prevents drip in lightly brined fish muscle by causing the proteins to swell and to bind liquid firmly, and not by a surface sealing action. In whole and in comminuted muscle at its natural pH, free drip is almost completely inhibited, and expressible drip greatly reduced, by incorporation of about 1 per cent NaCl. On either side of an approximate "isoelectric zone" (about pH 4.5 to 6.0) addition of HCl or NaOH causes fish muscle to swell markedly and to withhold liquid against hydraulic pressure, peptization of the proteins occurring above and below about pH 10 and 2 respectively. Between about pH 4.5 and 7.0, from 1 to 3 per cent NaCl enhances swelling and liquid binding power of fish muscle, while below pH 4.5 it has an entirely opposite effect. Addition of NaCl effects an increase in pH of fish muscle below, and a decrease above about pH 4.5. In 1 per cent NaCl fish muscle does not swell appreciably; in 3 to 7 per cent swelling is at a maximum, while higher concentrations cause an apparent decrease in swelling which may be due in part to the peptizing action of the salt. Theoretical and applied aspects of the process of brining fillets are discussed.

The isoelectric point of serum-globulin as determined by kataphoresis

Following the determination of the isoelectric point of insulin by kataphoretic methods (Howitt and Prideaux, 1932) it appeared to the authors that a similar determination for serum-globulin would be of interest owing to the globulin-like character of insulin. A comparison between the kataphoretic behaviours of these two proteins which are physiologically so different would also be afforded. Previous determinations of the isoelectric point of serum-globulin have generally been based on methods of maximum insolubility, or coagulation. Thus Kugelmass (1922) obtained a value of p H 4·55, whilst Krebs (1925), employing the coagulative action of proteins on a gold sol, found p H 5·4. The experiments of Rona and Michaelis (1910, 1927) gave the value 5·44 in close agreement with that of Krebs. Reiner (1927), however, has stated that serumglobulin has a broad isoelectric zone and is not a homogeneous substance. The findings of this worker are probably explained by the conclusions of Svcdberg and Sjögren (1928) that serum-globulin is a chemical entity, which degradated into fractions during normal treatment, such as “salting-out” with ammonium sulphate.