Electrophoretic concentration of macromolecules

An apparatus for concentrating macromolecules and removing macro molecules from nonionic solutes electrophoretically has been developed. Typical applications and techniques are outlined. Concentrations from 24- to 50-fold were achieved in a short period of time. The nondisruptive nature and versatility of the system is shown by the concentration of bovine serum albumin and three different enzymes.     

Concentration of dilute solutions of macromolecules by electrophoresis

An electrophoresis apparatus which is used for concentrating micrograms of macromolecules from solutions as large as 250 ml is described. The recoveries were greater than 91% with three different macromolecules tested (28 to 360 kDa). Solutions with volumes in the range of 35 ml were concentrated 70-fold in less than 90 min to a final volume of 0.5 ml. Larger volumes in the range of 250 ml were concentrated 227-fold in 16 h to a final volume of 1.1 ml. Sterile concentrates can be obtained if the apparatus is constructed under sterile conditions.     

Kerr effect of charged dipolar macromolecules without condensed counterions in conducting solution.

The theoretical treatment of the Kerr constant of rigid, dipolar, conducting ellipsoidal macromolecules of O'Konski and Krause (1970. J. Phys. Chem. 74:3243) has been extended to very low ionic strength solutions for charged macromolecules. The O'Konski and Krause theoretical treatment postulated a surface conductivity directly on the surface of each macromolecule. For charged macromolecules, this surface conductivity was generally assumed to be caused by movement of condensed counterions on the macromolecules. In the present work, it has been assumed that, at very low ionic strength, the average counterion is at the Debye characteristic distance from the surface of each charged macromolecule and contributes to surface conductivity at that distance, with no additional surface conductivity on the true surface of the macromolecule. Essentially, these considerations change the calculated interaction energy of the macromolecule with an externally applied electric field via a change in both the internal field components and in the reaction field of the macromolecular dipole. The new interaction energy is used to calculate the orientation distribution function of the macromolecules in solution and this distribution function can, in principle, be used to calculate the steady state electric linear or circular dichroism, electric light scattering, anisotropy of conductivity, etc., using the appropriate theoretical treatment for each of these quantities.     

Concentration of Virus from Water by Electro-Osmosis and Forced-Flow Electrophoresis. II. Improvement of Methodology and Application to Tap Water

The ability of the Canalco Model CF-3 electro-osmosis (EO) apparatus to concentrate viruses from artificially seeded distilled water was improved. Modification of the physical arrangement of the equipment allowed for a 10–25 fold increase in concentration efficiency and a concomitant decrease in the process time. The major improvements involved modifications of the cell arrangement (which increased the membrane transport area), a change in the salt replenishing solution and the use of different membranes of higher flux. Viruses concentrated by E0 from seeded tap water resulted in lower recoveries when compared to distilled water. The lower yields were probably due to instability or aggregation of the agents in the menstruum and not directly related to the physical apparatus. Under the conditions used, one could detect virus at levels as low as 0.01 plaque forming units (PFU) per ml of initial input. The efficacy of a modification of the Canalco forced-flow electrophoretic (FFE) system was also evaluated. The maximum potential was applied with a constant value for pump rates. A 6-fold concentration of virus and a 12-fold decrease in water volume was obtained.     

Contribution of the Excluded Volume of Bovine Serum Albumin for Solute Molecules to the Apparent Nonsolvent Water

Addition of a macromolecule to a solution will give rise to a large excluded volume for the centers of the solute molecules. This will cause an apparent increase in solute concentration which is of the same order of magnitude as that associated with the nonsolvent volumes reported in the literature. A critical examination of one of the procedures used for the determination of nonsolvent water—the vapor pressure method of Hill—is given, and it is concluded that, with the use of this method, it is impossible to detect any significant nonsolvent water surrounding bovine albumin for either sugars or polyols. Generally, data reported in the literature for the nonsolvent water of proteins or other macromolecules will be too high unless they are corrected for the excluded volume.     

A new interferometric technique for the determination of the electrophoretic mobility of macromolecules.

A novel device is described for measuring the electrophoretic mobility of monodisperse macromolecules. This apparatus, which is based upon a Twyman-Green interferometer, yields ±3% precision with runs lasting only 15 min; and it is suitable for temperatures and/or ionic strengths near or below physiological. Its operation is illustrated with bovine serum albumin, the apparent mobility of which is shown to vary strongly with the purity and concentration of the preparation.     

Crystallization and preliminary x-ray investigation of soybean beta-amylase.

Beta-Amylase [1, 4-alpha-D-glucan maltohydrolase, EC 3.2.1.2] has been purified from defatted soybean meal by fractional precipation with ammonium sulfate, ion-exchange chromatography on CM- and DEAE-Sephadex and gel filtration chromatography on Sephadex G-100. Two different components of beta-amylase were crystallized from ammonium sulfate solutions, and the homogeneity of each preparation was confirmed by sedimentation and disc electrophoretic analyses. Both components of soybean beta-amylase formed large single crystals (trigonal crystal system) from 40--50 per cent saturated ammonium sulfate solution buffered at pH 5.4 on dialyzing concentrated protein solution in the apparatus of Zeppezauer et al. Preliminary X-ray diffraction data gave a hexagonal lattice with unit cell dimensions a=86.1 A and c=144.4 A. The space group corresponds to P3121 or P3221, and one asymmetric unit contains one molecule of beta-amylase, assuming a crystal density of 1.25 g/ml and a molecular weight of the enzyme of 60,000 daltons. In this case, the crystal has a volume of 2.53 A-3 per atomic mass unit, and the percentage of protein in the crystal is about 52.     

Determination of vigabatrin by capillary electrophoresis with laser-induced fluorescence detection

A new analytical method for vigabatrin based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. 5-Carboxytetramethylrhodamine succinimidyl ester was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH9.5) containing 10 mM sodium dodecyl sulfate and a green He-Ne laser (excitation at 543.5 nm, emission at 589 nm). The concentration limit of detection in aqueous solution was 24 nM. Combined with a simple cleanup procedure, this method can be applied to the determination of vigabatrin in human plasma. A calibration curve ranging from 1.5 to 200 microM shown to be linear. Both the within-day and day-to-day reproducibilities and accuracies were less then 14.3% and 4.9% respectively. The limit of detection of vigabatrin in plasma was about 0.13 microM     

Removal of a bound ligand from a macromolecule by gel filtration.

If a solution of macromolecule of concentration c and volume a, initially saturated with a ligand of dissociation constant K, is gel filtered through a volume v of solution initially free of ligand, the saturation y with which it emerges is given by the equation y-lny-1=Kv/ca.     

Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature

An efficient optimization method for the crystallization of biological macromolecules has been developed and tested. This builds on a successful high-throughput technique for the determination of initial crystallization conditions. The optimization method takes an initial condition identified through screening and then varies the concentration of the macromolecule, precipitant, and the growth temperature in a systematic manner. The amount of sample and number of steps is minimized and no biochemical reformulation is required. In the current application a robotic liquid handling system enables high-throughput use, but the technique can easily be adapted in a nonautomated setting. This method has been applied successfully for the rapid optimization of crystallization conditions in nine representative cases.     

Fluctuation and linkage relations in macromolecular solution

It is shown in the appendix that the derivatives of the excess free energy of a macromolecule in solution, with respect to the activities of other solution components, lead to fluctuation and linkage relations among these other components. Solution fluctuation theory is used, but it is specialized to the fluctuations and correlations associated with the presence of a macromolecule, and is developed with a modified ensemble. The relations of the appendix are used to analyze the interaction of two solution components, A and B, with the macromolecule and with one another. Three cases are considered: (1) A and B are ligands that bind stoichiometrically to the macromolecule. This case reduces to Wyman's binding polynomial analysis. (2) A and B are two substances at high concentration that interact selectively with the macromolecule. (3) A is a species that binds stoichiometrically to the macromolecule, while B is a component at high concentration that interacts weakly with the macromolecule.     

108 Individual-particle electron tomography: a method for studying macromolecule dynamics

In solution, macromolecules are naturally flexible and dynamic. Dynamic personalities and structural heterogeneities of macromolecules are essential to understanding their proper function (Karplus & Kuriyan, 2005). However, structural determination of dynamic/heterogeneous macromolecules is limited by current technology such as: X-ray crystallography, nuclear magnetic resonance spectrum, small angle scattering, and electron microscopy single-particle reconstruction. A common weakness of all current techniques is requiring an averaged signal from thousands to millions of different macromolecules. Using averaged “signal” must involve in an assumption that macromolecules remain in identical structures or few identical conformations. This assumption is a good estimate for some macromolecule that have a rigid body, but not for most macromolecules that have “soft”, flexible, and dynamic body, such as lipoproteins and antibodies. An ideal approach for structure determination regardless of macromolecular dynamics is to use non-averaged signal, i.e. the signal from a single macromolecule itself. We developed a ‘‘focused electron tomography reconstruction’’ (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance macromolecule (Zhang & Ren, 2012). FETR can tolerate certain levels of image distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. Since this approach can obtain the structure of a single-instance macromolecule, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single ‘‘snapshot’’ of molecule structure determination (Zhang & Ren, 2012). FETR/IPET provides a completely new opportunity for a single-macromolecule structure determination, and could be used to study the dynamic character, equilibrium fluctuation, to reveal macromolecular mechanism, and even to track the intermediate state of the reaction of macromolecules (Zhang et al., 2010; Zhang & Ren, 2010).     

Activated carbons prepared from phosphoric acid activation of grain sorghum.

The production of activated carbons from grain sorghum with phosphoric acid activation has been studied by means of two processes, i.e., one-stage and two-stage. The former comprises simultaneous carbonization and activation after impregnation; the latter, the carbonization of the precursor at 300 degrees C for 15 min, followed by the activation of the resultant char after impregnation with phosphoric acid. The preparation conditions, e.g., activation duration, phosphoric acid concentration, and activation temperature, have been varied to determine the optimal processing conditions. The optimal activation conditions for the highest surface areas have been determined to be 600 and 500 degrees C with a phosphoric acid concentration of 35% for the one-stage and two-stage processes, respectively. The two-stage process has been found to greatly enhance the porosity development, especially the microporosity.     

Laser Doppler spectroscopy as applied to electrophoresis in protein solutions

The application of Laser Doppler spectroscopy (LDS) to the electrophoretic migration of macromolecules in solution by heterodyne light beating technique, previously developed by Ware and Flygare, has been improved by the design of a new microelectrophoresis cell and a high resolution in the frequency power spectrum. A 1024-channel correlator was used in combination with a software-controlled Fast Fourier transformation (FFT).This technique has been applied to single protein solution, bovine serum albumin (BSA), and to multicomponent systems, in particular to human blood serum. In comparison to normal free electrophoresis, LDS is more convenient and reveals more information in a much shorter period of time.     

Computer simulation as a tool for tracing the conformational differences between proteins in solution and in the crystalline state

Knowledge about the architecture of macromolecules has been derived primarily from crystallography. Therefore, it has been a matter of concern whether the conformation of a macromolecule in solution, namely in vivo, might be different from that in the crystalline state. To determine the difference between the conformations, a protein (trypsin inhibitor) dissolved in water has been simulated using the method of molecular dynamics and the results are compared with those obtained from a simulation of the full crystalline unit cell. We report here that no significant difference was found for backbone atoms, except for two more or less flexible loops extending from the core of the protein and the very flexible carboxyterminal residues. The side-chains in which the conformation in solution differs considerably from that in the crystal all belong to polar residues.     

A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules

The recently developed technique of two-dimensional (2D) cross-relaxation spectroscopy is utilized for systematic measurements of selective nuclear Overhauser enhancements (NOE) in the high resolution ¹H nuclear magnetic resonance (NMR) spectra of biological macromolecules in solution. Compared to conventional one-dimensional NOE studies, the 2D NOE experiment has the principal advantage that it avoids detrimental effects arising from the limited selectivity of preirradiation in crowded spectral regions. Furthermore, it yields with a single instrument setting a complete network of NOE's between all the protons in the macromolecule. The resulting information on intramolecular proton-proton distances provides a new avenue for studies of the spatial structures of biopolymers.     

Photobiotin as a sensitive probe for protein labeling

A sensitive method for the nonisotopic in vitro labeling of proteins under physiological conditions using photobiotin, a compound originally developed for labeling nucleic acids (Forster et al. (1985) Nucleic Acids Res. 13, 745), has been developed. Using sheep brain tubulin as a model protein it was shown that labeling with photobiotin resulted in detection limits below 10 pg when avidin-alkaline phosphatase was used in the final step. No significant loss of tubulin polymerization, colchicine binding, recognition by antitubulin antibodies, or changes in electrophoretic behavior were observed. In addition, photobiotinylation of antitubulin antibodies did not affect their recognition of unlabeled tubulin. The use of photobiotin labeling with avidin-alkaline phosphatase detection for electrophoretic separations of molecular weight standards, crude protein supernatants, and tubulin gave a 64 to 1024-fold increase in sensitivity over Coomassie blue staining.     

A Simple and Rapid Method for the Purification of Ovine Pineal Arylalkylamine N-Acetyltransferase

A two-step chromatographic procedure has been developed for the purification of ovine pineal arylalkylamine N-acetyltransferase (EC 2.3.1.87), based on the principles of disulfide exchange and anion exchange. The enzyme from 20 ovine pineal glands can be purified about 500-fold in a day; recovery is about 5%. Polyacrylamide gel electrophoretic analysis of the final preparation shows four major bands; one appears to be arylalkylamine N-acetyltransferase.     

Reproducibility of the biosynthetic parameters in various fermentation equipment

The final parameters of tetracycline biosynthesis in different fermentation apparatus were analysed comparatively with an account of possible changing of the fermentation broth volume against the initial one due to water evaporation. It was shown that for comparison of the biosynthesis parameters in different fermentation apparatus it is necessary to reestimate the activity values or the antibiotic concentration in a unit of the medium volume with respect to the initial volume of the fermentation medium charged into the apparatus.     

Trypsin-SBTI interaction in reverse micelles. A slow intermicellar exchange-dependent binding

Solubilisate exchange between reverse micelles must take place before any reaction inside reverse micelles occurs if the reactants are confined to the aqueous micellar core. When the interacting species are 2 small molecules or one small molecule and one macromolecule, it has been shown that the exchange is faster than the typical turnover of an enzymatic reaction. The study of the interaction between 2 macromolecules (trypsin and soybean trypsin inhibitor) in reverse micelles carried out in this work reveals that the exchange between these macromolecule-containing reverse micelles slows down by a thousand times and the limiting-step in the exchange, the fusion, by 10(6) times. Both reverse micellar size (omega 0 = [water]/[surfactant]) and temperature affected the rate of the fusion process. A hypothesis for the proposed active role of macromolecules in the exchange process is also given.