Binding of ampholine to transfer RNA.

The melting temperature of isoaccepting tRNAfMet is affected by Ampholine. The plot of Tm versus the logarithm of Ampholine concentration shows clearly an increasing effect of Ampholine when the pH changes from 7.4 to 4.2. This result is interpreted as binding of Ampholine to the nucleic acid. The effects of Ampholine have been compared with those of soidum, magnesium and tetraethylene pentamine. Ampholine carrier ampholytes at pH 4.2 bind to tRNA with the same affinity as magnesium; at higher pH values they are less active. An hypothesis for the mechanism of action of Ampholine on nucleic acids during isoelectric focusing is proposed.     

The role of surface charge on the accelerating action of heparin on the antithrombin III-inhibited activity of alpha-thrombin

We have compared surface charge and the surface charge density on the polyanions heparin and potassium polyvinyl sulfate (KPVS), as well as on hydrolyzed heparin and KPVS, with their accelerating effect on the inhibitory action of antithrombin III on thrombin. Polyelectrolyte titration of thrombin with KPVS or heparin at pH 7.4 clearly indicates an electrostatic interaction. In contrast, at the same pH no electrostatic interaction is observed between polyanions and antithrombin III. KPVS accelerates the inhibitory action of antithrombin III to the same extent as heparin on the basis of charge equivalence. Heparin and KPVS with a mean distance between two charged centers of less than 0.75 and 0.95 nm, respectively, accelerate strongly whereas hydrolysates with lower charge densities are far less active. The following observations are indicated. Intramolecular neutralization of oppositely charged residues occurs within thrombin, antithrombin III, and partially hydrolyzed heparin. Heparin acts on the antithrombin III-thrombin reaction through cooperative electrostatic binding to thrombin and nonelectrostatic interaction with antithrombin III. This indicates a quasi-catalytic action of the polyelectrolyte. Hydrolysis of only a few N-sulfate residues within the heparin molecule decreases the linear surface charge density to such an extent that the accelerating action is drastically reduced. The loss of accelerating capacity agrees with the sudden loss of counterion condensation due to the decrease of the linear surface charge density beyond limits postulated by Manning in a theory of polyelectrolytes.     

Heparin binding is necessary, but not sufficient, for fibronectin aggregation. A fluorescence polarization study

Analysis of parameters governing heparin binding to fibronectin indicates that heparin binding is a necessary, but insufficient, condition for fibronectin cryoprecipitation. Heparin binding to fibronectin is a rapid, readily reversible event which can occur under several conditions which prohibit fibronectin cryoprecipitation. While cryoprecipitation of fibronectin is abolished at temperatures in excess of 10 degrees C, appreciable heparin binding to fibronectin does occur even at 40 degrees C. While increasing ionic strength and pH inhibit both heparin binding and cryoprecipitation of fibronectin, heparin binding can still occur at high ionic strengths and pH values which completely abolish cryoprecipitation. Scatchard analysis of fluorescent polarization data reveals a biphasic heparin binding curve with high and low affinity Kd values of 3.5 X 10(-8) and 10(-6) M, respectively. In contrast to heparin binding, fibronectin aggregation is a cooperative phenomenon. Fibronectin cryoprecipitation is greatly reduced at temperatures above 10 degrees C, at pH values above pH 10, and at ionic strengths above 0.3 M. Thus, heparin binding and protein aggregation are separate events which occur during fibronectin cryoprecipitation. Results obtained here via fluorescence polarization in conjunction with other physical measurements suggest that a decrease in flexibility of the fibronectin molecule is associated with the protein aggregation step of cryoprecipitation. The role of heparin in the mechanism of fibronectin cryoprecipitation is discussed.     

Anomalous behaviour of yeast isocitrate dehydrogenase during isoelectric focusing

Isoelectric focusing of yeast isocitrate dehydrogenase apparently reveals a number of ;isoenzymes'. These have isoelectric points near pH5.5 in crude material, but during purification the mean isoelectric point progressively rises to pH7.0 and the band pattern changes. The shift in isoelectric point during purification is apparently genuine, since it is also manifested in the electrophoretic and chromatographic properties of the enzyme. The multiple forms, however, are an artifact, generated by exposure of the enzyme to Ampholine, since their activities vary with the protein/Ampholine ratio and they cannot be observed in any system from which Ampholine is excluded. There are no detectable isoenzymes of yeast isocitrate dehydrogenase.     

Studies on pig serum lipoproteins. VI. Surface charge of very low density lipoprotein.

The surface electric charge of pig serum very low density lipoprotein (VLDL) is described. By isoelectric focusing VLDL was separated into at least 3 fractions having different isoelectric points and polypeptide distributions. The ultracentrifugal and electron microscopic results indicate that the VLDL was not drastically denatured by Ampholine.     

Multiple roles of heparin in the aggregation of p25α

The 219-residue protein p25α stimulates the fibrillation of α-synuclein (αSN) in vitro and colocalizes with it in several α-synucleinopathies. Although p25α does not fibrillate by itself under native conditions in vitro, αSN-free p25α aggregates have also been observed in vivo in, for example, multiple system atrophy. To investigate which environmental conditions might trigger this aggregation, we investigated the effect of polyanionic biomolecules on p25α aggregation. Heparin, polyglutamate, arachidonic acid micelles, and RNA all induce p25α aggregation. More detailed studies using heparin as template for aggregation reveal that a minimum of 10-14 heparin monosaccharide units per heparin polymer are required. Bona fide fibrils are only formed at intermediate heparin concentrations, possibly because an excess of heparin binding sites blocks the inter-p25α contacts required for amyloid formation. Other polyanions also show an optimum for amyloid formation. Aggregation involves only modest structural changes according to both spectroscopic and proteolytic experiments. The aggregates do not seed aggregation of heparin-free p25α, suggesting that heparin is required in stoichiometric amounts to form organized structures. We are able to reproduce these observations in a model involving two levels of binding of p25α to heparin. We conclude that the modest structural changes that p25α undergoes can promote weak intermolecular contacts and that polyanions such as heparin play a central role in stabilizing these aggregates but in multiple ways, leading to different types of aggregates. This highlights the role of non-protein components in promoting protein aggregation in vivo.     

Suppression of insulin aggregation by heparin

The aggregation of insulin near its isoelectric point and at low ionic strength was suppressed in the presence of heparin. To understand this effect, we used turbidimetry and stopped-flow to study the pH- and ionic strength ( I)-dependence of the aggregation of heparin-free insulin. The results supported the role of interprotein electrostatic interactions, contrary to the commonly held view that such forces are minimized at pH = pI. Electrostatic modeling of insulin (DelPhi) revealed that attractive interactions arise from the marked charge anisotropy of insulin near pI. We show how screening of the interprotein attractions by added salt lead to maximum aggregation near I = 0.01 M, corresponding to a Debye length nearly equal to the diameter of the insulin dimer, consistent with a dipole-like protein charge distribution. This analysis is also consistent with suppression of aggregation by heparin, a strong polyanion that by binding to the positive domain of one protein, inhibits its interaction with the negative domain of another.     

Nonisoelectric focusing in buffers.

Stable pH gradients were formed and focusing of proteins was carried out in polyacrylamide gels containing mixtures of simple, amphoteric buffers, replacing the Ampholine hitherto used in isoelectric focusing (IF). Stable pH gradients can also be formed between acid anolyte and basic catholyte if Ampholine is replaced by nonamphoteric buffers. The fact that focusing can be carried out with nonampholytes shows that focusing in this case is, and in all other cases may be, nonisoelectric. It is postulated that the pH gradient in IF forms by steady-state stacking (isotachophoresis) and forms within the stack. In distinction to ordinary steady-state stacking, however, the stack remains confined within the gel (or density gradient) since the strong acid and base in the electrolyte reservoirs bar by deprotonation or electrostatic repulsion migration into the electrode chambers.     

Structural characterization and the determination of negative cooperativity in the tight binding of 2-carboxyarabinitol bisphosphate to higher plant ribulose bisphosphate carboxylase

When CO2/Mg2+-activated spinach leaf ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) is incubated with the transition-state analog 2-carboxyarabinitol 1,5-bisphosphate, an essentially irreversible complex is formed. The extreme stability of this quaternary complex has allowed the use of native analytical isoelectric focusing, anion-exchange chromatography, and nondenaturing polyacrylamide gel electrophoresis to probe the mechanism of the binding process and the effects of ligand tight-binding on the structure of the protein molecule. Changes in the chromatographic and electrophoretic properties of the enzyme upon tight binding of the inhibitor reveal that the ligand induces a conformational reorganization which extends to the surface of the protein molecule and, at saturation, results in a 16% decrease in apparent molecular weight. Analysis of ligand binding by isoelectric focusing shows that (i) incubating the protein with a stoichiometric molar concentration of ligand (site basis) results in an apparently charge homogeneous enzyme population with an isoelectric point of 4.9, and (ii) substoichiometric levels of ligand produce differential effects on each of the charge microheterogeneous native enzyme forms. These stoichiometry-dependent changes in electrofocusing band patterns were employed as a probe of cooperativity in the ligand tight-binding process. The tight-binding reaction was shown to be negatively cooperative.     

Purification of cat submaxillary kallikrein.

The cat submaxillary gland contains 1,000--2,400 kallikrein units (KU)/g of tissue. The submaxillary kallikrein was purified to homogeneity by acetone fractionation, DEAE-Sephadex A-50 chromatography, Sephadex G-75 gel filtration, and Ampholine isoelectric focusing. The kallikrein was separated by isoelectric focusing into 6--7 forms with pI's between 4.2 and 5.1. One mg of the purified kallikrein contained 930--1,260 KU in the dog vasodilator assay, and hydrolyzed 15--25 and 9--12 mumol of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and N-alpha-toluenesulfonyl-L-arginine methyl ester (TAME), respectively, in 1 min at 25 degrees C and pH 8.0. The Km's of the purified kallikrein with BAEE and TAME were 0.67 and 0.34 mM, respectively. Hydrolysis of N-alpha-benzoyl-L-tyrosine ethyl ester (BTEE), N-alpha-benzoylarginine-p-nitroanilide (BApNA), and casein was small or negligible. The apparent molecular weight of the kallikrein was estimated to be 5 X 10(4) by Sephadex G-100 gel filtration and 4.7 X 10(4) by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). The kallikrein was found to contain 18.5% carbohydrate by weight. Trasylol and soybean trypsin inhibitor were not specific inhibitors of this kallikrein.     

Comparison of malate dehydrogenase isoenzymes in someAllium species by isoelectric focusing

Malate dehydrogenase isoenzymes were studied in tenAllium species and in six cultivars ofA. cepa by isoelectric focusing in polyacrylamide gel with Ampholine pH 3.5–10.0. Using this method better resolution was obtained than by polyacrylamide gel electrophoresis. The number of MDH isoenzymes obtained by isoelectric focusing is from five to ten in the range of pH 3.65 to 6.75. MDH isoenzymes can be used for characterization on the level of species and cultivars (inA. cepa), but its use on the level of sections and subgenera is questionable.     

Precipitation of egg white proteins below their isoelectric points by sodium dodecyl sulphate and temperature.

The precipitating of effect of sodium dodecyl sulphate (SDS) on the egg white proteins ovalbumin, conalbumin and lysozyme was studied at 25 degrees C and at different pH values. The proteins precipitated below their respective isolectric points, provided the detergent to protein ratio was appropriate. The pH profile of precipitation was different for the three proteins reflecting net charge differences. The binding of SDS to the proteins was studied with [35S]-labelled SDS and for ovalbumin a ratio of 21--28 SDS molecules/protein molecule, dependent on the concentration of SDS initially used, seem to be required for precipitation at pH 4.5. This number, however, is dependent on pH and increases with an increased positive net charge of the protein. The precipitating effect of SDS was identical for ovalbumin, conalbumin and lysozyme when compared on a gram to gram basis (0.1--0.15 g SDS/g precipitated protein). The precipitated protein was denatured as measured by differential scanning calorimetry, but was also completely redissolved if pH was increased to above the isoelectric point. The precipitating effecto f SDS was also examined at elevated temperatures. The two-phase systems of the proteins induced by SDS at 25 degrees C were heated from 25 degrees C to 90 degrees C at a rate of 1.25 degrees C/min. The precipitation behaviour was similar for the three proteins upon heating. When the SDS concentration was increased the precipitation curves were transferred towards lower temperatures and the courses of precipitation became less sharp. The synergistic effect of SDS and heat on protein precipitation was differentiated by denaturation measurements and radioactive labelling. The ratio SDS to precipitated protein gradually diminished towards higher temperatures but no purely thermal precipitation was found.     

Preparative isoelectric focusing of human serum very low-density and low-density lipoproteins.

Ten percent glycerol prevented the usual precipitation of human serum very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) at their isoelectric points during their preparative isoelectric focusing (IEF), IEF separated VLDL and LDL into two major fractions. The observed optical density peaks are not artifacts caused by binding of Ampholines to VLDL or LDL since no radioactivity accumulated in the fractions containing VLDL or LDL during IEF in the presence of [¹⁴C]Ampholine, and gel filtration completely separated the lipoproteins from [¹⁴C]Ampholine. These results suggest that IEF may separate subspecies of VLDL and LDL under suitable experimental conditions.     

Nature and significance of the interactions between amyloid fibrils and biological polyelectrolytes

Charged polyelectrolytes such as glycosaminoglycans and nucleic acids have frequently been found associated with the proteinaceous deposits in the tissues of patients with amyloid diseases. We have investigated the nature and generality of this phenomenon by studying the ability of different polyanions, including DNA, ATP, heparin, and heparan sulfate, to promote the aggregation of amyloidogenic proteins and to bind to the resulting aggregates. Preformed amyloid fibrils of human muscle acylphosphatase and human lysozyme, proteins with a net positive charge at physiological pH values, were found to bind tightly to the negatively charged DNA or ATP. The effects of the polyelectrolytes on the kinetics of aggregation were studied for acylphosphatase, and the presence of ATP, DNA, or heparin was found to increase its aggregation rate dramatically, with a degree dependent on the net charge and size of the polyanion. Magnesium or calcium ions were found to attenuate, and ultimately to suppress, these interactions, suggesting that they are electrostatic in nature. Moreover, heparin was found to stabilize the aggregated state of acylphosphatase through compensation of electrostatic repulsion. Noteworthy, differences in affinity between native and aggregated acylphosphatase with heparin suggest that amyloid fibrils can themselves behave as polyelectrolytes, interacting very strongly with other polyelectrolytes bearing the opposite charge. Within an in vivo context, the strengthening of the electrostatic interactions with other biological polyelectrolytes, as a consequence of protein misfolding and aggregation, could therefore result in depletion of essential molecular components and contribute to the known cytotoxicity of amyloid fibrils and their precursors.     

Effect of heparin on protein aggregation: inhibition versus promotion

The effect of heparin on both native and denatured protein aggregation was investigated by turbidimetry and dynamic light scattering (DLS). Turbidimetric data show that heparin is capable of inhibiting and reversing the native aggregation of bovine serum albumin (BSA), β-lactoglobulin (BLG), and Zn-insulin at a pH near pI and at low ionic strength I; however, the results vary with regard to the range of pH, I, and protein-heparin stoichiometry required to achieve these effects. The kinetics of this process were studied to determine the mechanism by which interaction with heparin could result in inhibition or reversal of native protein aggregates. For each protein, the binding of heparin to distinctive intermediate aggregates formed at different times in the aggregation process dictates the outcome of complexation. This differential binding was explained by changes in the affinity of a given protein for heparin, partly due to the effects of protein charge anisotropy as visualized by electrostatic modeling. The heparin effect can be further extended to include inhibition of denaturing protein aggregation, as seen from the kinetics of BLG aggregation under conditions of thermally induced unfolding with and without heparin.     

Analytical isotachophoresis in capillary tubes. Analysis of hemoglobin, hemiglobin cyanide and isoelectric fractions of hemiglobin cyanide.

The zone stabilization in capillary isotachophoresis in the water phase has been improved by methylcellulose so that proteins can be analysed. Hemoglobin and hemiglobin cyanide samples were studied as model systems. Ampholine carrier ampholytes were used as spacers, enhancing the detection of the different components. The optimal amounts of Ampholine, however, were found to be much smaller than in most of the previously published reports. Linear relationships were found between the zone lengths and sample amounts, including spacers. The separations were reproducible and reached the isotachophoretic steady state. The hemiglobin cyanide was fractionated by isoelectric focusing. The four main fractions were then analyzed by capillary isotachophoresis and shown to be heterogeneous in mobility with a pH of 7.5 in the leading electrolyte. The component zones of the total hemiglobin cyanide sample were all identified in relation to the isotachophoretic components of the isoelectric fractions. The total analysis time was in average 30-40 min. The sample amounts were about 40 mug protein in each experiment with very small Ampholine volumes, 25-100 nl 40% (w/v).     

Parathyroid hormone is a heparin/polyanion binding protein: binding energetics and structure modification

The interaction of four representative polyanions with parathyroid hormone (PTH) residues 1-84 has been investigated utilizing a variety of spectroscopic and calorimetric techniques. Each of the polyanions employed demonstrate enthalpically driven binding to PTH (1-84) with significant affinity. The polyanions heparin, dextran sulfate, phytic acid, and sucrose octasulfate induce alpha-helical structure in PTH to varying extents depending on the ratio of polyanion to protein employed. Intrinsic and extrinsic fluorescence spectroscopy suggests significant protein tertiary structure alteration upon polyanion binding. Although structural modification occurred upon polyanion binding, PTH colloidal stability was increased depending on the ratio of polyanion to protein used. Nevertheless, the bioactivity of PTH in the presence of various ratios of heparin was not altered. The potential biological significance of PTH/polyanion interactions is discussed.     

Extent of charge screening in aqueous polysaccharide solutions

The absorption optical system of a Beckman XL-I ultracentrifuge has been used to monitor the Donnan distribution of ions in polysaccharide solutions dialyzed against sodium phosphate buffer (pH 6.8, I 0.08) supplemented with 0.2 mM chromate as an indicator ion. For dextran sulfate, heparin, and polygalacturonate, the effective net charges are shown to be only one-third of those deduced from the chemical structures--a reflection of charge screening (counterion condensation) in aqueous polyelectrolyte solutions. Whereas the extent of charge screening for the first two polysaccharides agrees well with theoretical prediction, the disparity in the corresponding comparison for polygalacturonate reflects partial esterification of carboxyl groups, whereupon the experimental parameter refers to the effective charge per hexose residue rather than the effective fractional charge of each carboxyl group.     

Purification and comparative properties of the glycoprotein nicotinamide adenine dinucleotide glycohydrolase from rat liver microsomal and plasma membranes.

NAD glycohydrolase, or NADase (NAD+ glycohydrolase, EC 3.2.2.5) was solubilized with porcine pancreatic lipase from isolated fractions of microsomes and plasma membranes obtained from rat livers. The enzyme from each organelle was further purified by DEAE-cellulose chromatography, gel filtration and isoelectric focusing. The solubilized, partially purified enzymes had similar molecular weights, pH-activity profiles and Km values. Marked charge heterogeneity was observed for the microsomal enzyme on isoelectric focusing between pH 6 and 8 with maximum activity focusing at pH 8.0. Plasma membrane NADase displayed a single peak at pH 6.7. Treatment of the partially purified microsomal or plasma membrane enzyme with neuraminidase resulted in a single peak of activity on isoelectric focusing (pH 3.5--10) with a pI of 9.2. Polyacrylamide gel electrophoresis of either NADase revealed a periodate-Schiff positive band which was coincident with enzyme activity. Compositional analyses of the microsomal enzyme focusing at pH 8.0 confirmed the presence of hexoses, hexosamines and sialic acid. Differences in carbohydrate composition might be important in determining the subcellular distribution of this enzyme.     

Use of amphiphilic spin labels and whole cell isoelectric focusing to assay charge characteristics of sperm surfaces.

The charge characteristics of the surface of bull and rabbit sperm were analyzed using surface-directed spin labels and whole cell isoelectric focusing. Spin label experiments tested charges believed to be localized at the membrane phospholipid-water interface. Charge properties of the glycoprotein calyx were analyzed with isoelectric focusing. Addition of charged detergents altered spin label spectra without changing the isoelectric focusing pH value. Sperm presumed to differ in the amount of adsorbed protein had different isoelectric focusing pH values, but similar spin label spectra. We conclude that these techniques are capable of monitoring charge domains on the sperm surface: one at the polar surface of the phospholipid membrane and one at the interface between the glycocalyx and the suspending fluid. Furthermore, changes in charge density are induced in unique zones of the cell surface during sperm maturation.