Continuous optical scanning in polyacrylamide gel electrophoresis:estimation of the apparent diffusion coefficient of beta-lactoglobulin B.

The continuous scanning apparatus developed by Catsimpoolas was applied to an analysis of the concentration profiles of a protein, β-lactoglobulin B, while it was subjected to polyacrylamide gel electrophoresis (PAGE) in a multiphasic buffer system. Continuous optical scanning in PAGE permitted reliable estimation of the standard deviation of the concentration profile (σ), the relationship between σ² and time, and the apparent diffusion coefficient, D′, derived from σ², as the current density varied from 2 to 9 mA/cm², protein load varied from 250 to 900 μg/cm², and the ionic strength varied from 0.015 to 0.065 m. Under these conditions, D′ was linearly related to current density and protein load. Further, log (D′) was linearly related to gel concentration (%T) ranging from 6 to 14%. However, D′ was nonlinearly related to ionic strength. Due primarily to the ionic strength factor, the apparent diffusion coefficient of protein in gels appeared to be approximately 10-fold larger than under the conditions of high ionic strength conventionally used in sedimentation and diffusion studies. Extrapolation of D′ to 0% T, zero protein load, zero current density, and “infinite” ionic strength (assuming noninteraction of these factors), as well as correction for viscosity and temperature, yielded an estimated free-diffusion coefficient, D_20,w, of 3.1 × 10^?7 cm²/s, which is compatible with previously reported values. These studies indicate that the optimal resolution obtained by PAGE will be considerably lower than that predicted theoretically on the basis of free-diffusion coefficients, and suggest that electrostatic interaction between the proteins and/or deformation of voltage gradient and pH within the protein zones may contribute significantly to band spreading.     

Transient state isoelectric focusing. Experimental determination of apparent diffusion coefficients in polyacrylamide gels

A photoelectric scanning assembly utilizing uv absorption optics and an on-line digital data acquisition and processing system has been used to follow kinetically zone spreading during the defocusing stage (absence of electric field) of transient state isoelectric focusing (TRANSIF) in polyacrylamide gels. Measurement of the variance (σ²) of a diffusing zone as a function of time yields a linear relationship, the slope of which corresponds to the apparent diffusion coefficient (D) of the protein. A linear relationship is also obtained when the logarithm of the apparent diffusion coefficients (logD) are plotted vs acrylamide concentration (T). This relationship can be used to extrapolate D to zero gel concentration. The apparent diffusion coefficient measured in this way is significantly larger than the true diffusion coefficient. The slope of the plot logD vs T, designated C_R, is expected to be a measure of molecular size related to the retardation coefficient in polyacrylamide gel electrophoresis.     

The effect of SDS on protein zone dispersion in polyacrylamide gel electrophoresis

The zone dispersions of the reduced subunit of β-lactoglobulin B and its derivative with sodium dodecyl sulfate (SDS) were measured during polyacrylamide gel electrophoresis (PAGE) using the apparatus for continuous optical scanning at 280 nm. The ratio of apparent diffusion coefficients (D′) of the reduced subunit of β-lactoglobulin B (1.71 × 10^?6 cm²/s) and of its SDS-derivative (7.1 × 10^?7 cm²/s) was found to be 2.4 under the conditions of PAGE (pH 10.4, 0.015 ionic strength, 1°C, 4 mA/cm² current density, 50 μg protein load, 10% T gel) used. This is nearly twice the value of 1.3 predicted, under the assumption of sphericity for these protein molecules, on the basis of the binding of 1.4 g of SDS per gram of protein. It is postulated that the increment in zone sharpness (decrease in apparent diffusion coefficient) over that predicted by SDS binding alone is a general property of SDS-proteins providing gel electrophoresis in SDS-containing buffers with a resolving power larger than that obtained in the absence of the detergent.     

Dynamic Light Scattering Application to Study Protein Interactions in Electrolyte Solutions

The concentration dependence of the diffusion coefficient of particles suspended in solution depends primarily on the occupied volume fraction and on repulsive and attractive forces. This dependency is expressed by the interaction parameter, which can be assessed experimentally by light scattering measurements and have been determined for the diffusion coefficient of BSA under different salt concentration conditions in the present work. The result shows that the diffusion coefficient of protein grows up with increasing protein concentration, and when the ionic strength turns up gradually the diffusion coefficient decreases with protein concentrations increasing. The concentration dependence of BSA diffusion coefficients is interpreted in the context of a two-body potential of mean force, which includes repulsive hard-sphere and Coulombic interactions and attractive dispersion. With the increase of ionic strength, Debye screening decreases, protein interaction changes from repulsion to attraction, and protein begins to aggregate. By means of the concentration dependence of BSA diffusion coefficients, one can obtain the parameters of protein interactions and can find that protein bears a net effective charge of –9.0 e and has a Hamaker constant of 2.8k_BT. This work demonstrates that DLS is an effective technique of studying protein interactions.     

Inelastic light scattering studies of solutions of the chloroplast coupling factor (CF1) at high and low ionic strength

The translational diffusion coefficient of CF₁ at low and high protein concentration as well as at different ionic strength (0.05 – 1.65 M) wsa determined by means of quasi-elastic light scattering experiments. The diffusion coefficient changes from D_20,w^o = 3.12 × 10^?7 cm² · sec^?1 at 0.05 M, pH 7.8, 20°C, to D_20,w^o = 3.52 × 10^?7 cm² · sec^?1 at 1.6 M, pH 7.8, 20°C. At high enzyme concentration (20 mg/ml) and under crystallization conditions (Paradies, BBRC 91: 685, 1979) CF₁ behaves as a solution of “true” hard spheres, whereas at low salt concentration the ionic atmosphere has a larger spatial extent, resulting in a higher effective hydrodynamic radius (R_H = 65 Å).     

Transient state isoelectric focusing: effects of zone load and carrier ampholyte concentration on the kinetics of defocusing and refocusing in sucrose density gradients

The kinetics of focusing, defocusing, and refocusing of l-histidyl-l-tyrosine in a sucrose density gradient have been studied utilizing a special apparatus for repetitive scanning of the isoelectric focusing column, employing uv absorption optics and a digital data acquisition system. Starting from a uniform or triangular concentration profile, the band is “focused” and a nearly linear pH gradient is formed during initial focusing. The electrical field is then abolished and free diffusion occurs. The electrical field is then reapplied (“refocusing”) and the band is allowed to sharpen, presumably reapproaching a steady state. The band width was measured quantitatively as the second moment about the mean (square of the standard deviation, σ²). In theory, measurements of σ² versus time permit the estimation of the apparent diffusion coefficient (D) and the isoelectric focusing parameter (pE). If the electrical field strength E and the pH gradient, $\text{d(}\text{pH}\text{)}\text{dx}$, were also measured, then one could calculate the slope of the pH mobility curve of the protein $\text{dM}\text{d(}\text{pH}\text{)}$ evaluated at the isoelectric point. D can be measured during the defocusing stage, and $\text{pE,}\text{D}\text{pE}\text{,}\text{or}\text{D}$ can be measured during focusing or refocusing. Several limitations and difficulties in the verification of this theory have been encountered: First, the apparent diffusion coefficient depends on zone load in approximately a linear fashion. Accordingly, it is necessary to measure D at several zone loads, and then extrapolate to zero load by linear regression techniques. Second, the ampholyte concentration has a marked effect on both D and pE. Here we have no a priori reason to extrapolate to zero ampholyte concentration. Also, at present we have no satisfactory method for measurement of E. These preliminary studies should be helpful in indicating further directions for experimental refinement and for generalization of theory.     

QELS-SEF study on poly(lysine) in the extraordinary phase: Effect of electric field strength on the apparent diffusion coefficient

The technique of quasielastic light scattering (QELS) in the presence of a sinusoidal electric field (QELS-SEF) was used to examine the diffusion of poly(lysine) in the extraordinary phase, which is defined by the anomalously small value in the apparent diffusion coefficient (D_app, obtained by QELS methods) as the added salt concentration is lowered below 10 mM. Spectra were obtained for high-molecular-weight poly(lysine) (800,000) in 0.5 mM KCl, using a driving frequency of 90 Hz. It was observed that the linewidth, hence apparent diffusion coefficient (D_SF), increased with electric field strength (E) over the range 6 < E < 30 V/cm. As might be anticipated, the asymptotic limit, D_SF(E = 0), was almost numerically equivalent to D_app at this KCl concentration. As E approached the upper limit value of 31 V/cm, the value of D_SF approached the high salt (500 mM KCl) value of D_app. These observations are discussed in terms of small-ion dispersion effects.     

Influence of λ-DNA concentration on mobilities and dispersion coefficients during agarose gel electrophoresis

Earlier works demonstrated theoretically and experimentally that during gel electrophoresis the mobility μ and the dispersion coefficient D_x [reflecting band broadening; G. W. Slater and J. Noolandi (1985) Physics Review Letters, Vol. 55, pp. 1579–1582; T. A. Duke, A. N. Semenov, and J. L. Viovy (1992) Physics Review Letters, Vol. 69, pp. 3260–3263] depend on the chain length, the electric field, and on the gel concentration. Using a Fluorescence Recovery After Photobleaching setup coupled with an electrophoretic cell, we show that they also depend of the DNA concentrationC. Two regimes are observed. The first is analogous to a “dilute” regime in the gel where μ and D_x are DNA concentration independent. In the second regime, μ and D_x decrease when C increases. These results are explained by DNA-DNA interactions. As expected the C^* concentration, under which measurements must be carried out to avoid this effect, is found to be the same as the overlap concentration C^* determined in solution. Using concentrations of the studied DNA lower than its C^*, μ and D_x show a field dependence in good agreement with the predictions of the Biased Reptation model with Fluctuations. © 1997 John Wiley & Sons, Inc. Biopoly 42: 471–478, 1997     

Diffusion of insulin-like growth factor-I and ribonuclease through fibrin gels

A fluorescence-based method for simultaneously determining the diffusion coefficients of two proteins is described, and the diffusion coefficient of insulin-like growth factor (IGF-I) and ribonuclease (RNase) in a 0.27% fibrin hydrogel is reported. The method is based on two-color imaging of the relaxation of the protein concentration field with time and comparing the results with a transport model. The gel is confined in a thin (200 μm) capillary and the protein is labeled with a fluorescent dye. The experimentally determined diffusion coefficient of RNase (D = 1.21 × 10⁻⁶ cm²/s) agrees with literature values for dilute gels and bulk aqueous solutions, thus indicating the gel and the dye had a negligible effect on diffusion. The experimental diffusion coefficient of IGF-I (D = 1.59 × 10⁻⁶ cm²/s), in the absence of binding to the fibrin matrix, is consistent with the dimensions of the molecule known from x-ray crystallography and a correlation between D and molecular weight based on 14 other proteins. The experimental method developed here holds promise for determining molecular transport properties of biomolecules under a variety of conditions, for example, when the molecule adsorbs to the gel or is convected through the gel by fluid transport.     

Diffusion NMR-based comparison of electrostatic influences of DNA on various monovalent cations

Counterions are important constituents for the structure and function of nucleic acids. Using ⁷Li and ¹³³Cs nuclear magnetic resonance (NMR) spectroscopy, we investigated how ionic radii affect the behavior of counterions around DNA through diffusion measurements of Li⁺ and Cs⁺ ions around a 15-bp DNA duplex. Together with our previous data on ²³Na⁺ and ¹⁵NH₄⁺ ions around the same DNA under the same conditions, we were able to compare the dynamics of four different monovalent ions around DNA. From the apparent diffusion coefficients at varied concentrations of DNA, we determined the diffusion coefficients of these cations inside and outside the ion atmosphere around DNA (D_b and D_f, respectively). We also analyzed ionic competition with K⁺ ions for the ion atmosphere and assessed the relative affinities of these cations for DNA. Interestingly, all cations (i.e., Li⁺, Na⁺, NH₄⁺, and Cs⁺) analyzed by diffusion NMR spectroscopy exhibited nearly identical D_b/D_f ratios despite the differences in their ionic radii, relative affinities, and diffusion coefficients. These results, along with the theoretical relationship between diffusion and entropy, suggest that the entropy change due to the release of counterions from the ion atmosphere around DNA is also similar regardless of the monovalent ion types. These findings and the experimental diffusion data on the monovalent ions are useful for examination of computational models for electrostatic interactions or ion solvation.     

Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica

R-phycoerythrin is one of the three phycobiliproteins which are extensively employed as fluorescent probes, and it is prepared from red macroalgae. Phycobiliproteins in the marine red macroalga Heterosiphonia japonica were extracted in 50 mM phosphate buffer (pH 7.0) and precipitated by salting-out. The R-phycoerythrin was isolated by gel filtration with Sepharose CL-4B and Sephadex G-200. Then it was purified by ion exchange chromatography on DEAE Sepharose Fast Flow which was developed by linear ionic strength gradients. The purified R-phycoerythrin gave a ratio of A₅₆₅ to A₂₈₀ of 4.89. It showed a single band and a pI of 4.8 on the examination by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. The polypeptide analysis of the purified R-phycoerythrin by SDS–PAGE demonstrated that it contains four chromophore-carrying subunits and no colorless polypeptide and has two hexameric aggregates. The preparative procedures of the R-phycoerythrin purification established based on the experiments exhibit advantages and can offer a reference for R-phycoerythrin preparation from other marine red macroalga.     

Purification, characterization and reassembly of the bacteriophage T4D tail sheath protein P18.

P18, the sole component of T4 tail sheath, has been isolated in a monomeric active form from extended sheaths of intact tails which were dissociated at low ionic strength. The molecular weight of P18 is determined to be 65,000 from sedimentation equilibrium and 73,000 from sodium dodecyl sulphate/gel electrophoresis. Combining the diffusion constant (D_20,w = 5·5× 10^?7cm²s^?1)and the sedimentation constant (s⁰_20,w = 4·2 S) a value of 67,000 is obtained. The circular dichroism spectra reveal a striking similarity of the structure of P18 in the monomeric state and in the extended sheath conformation.The purified P18 is found to reassemble into extended sheaths if the core-baseplate complex is present, forming normal length tails. Structures similar to polysheath are formed in the absence of core-baseplates.     

Quasielastic light scattering by biopolymers. V. Interparticle interactions between polynucleosomes

Quasielastic light scattering and electrophoretic light scattering experiments were performed on chicken erythrocyte polynucleosome solutions at various temperatures and ionic strengths. The apparent diffusion coefficient, D_app, was found to depend on the scattering vector K. In general, D_app can be described as a damped oscillatory function of K in the ionic strength range of 10 to 60 mM and over the temperature range of 10 to 40°C. Electrophoretic light scattering studies on total digest chromatin samples indicate the apparent charge on the polynucleosomes increases as the ionic strength is lowered from 10 to 1 mM. These data are interpreted in terms of fluctuations in the surface charge distribution of the polyion and subsequent inducement of an asymmetric distribution of small ions about the polyion. These fluctuation components lead to the formation of “clusters” of polyions.     

The Molecular Weight and Other Biophysical Properties of Bromegrass Mosaic Virus

Data are presented which show that bromegrass mosaic virus has a particularly low molecular weight and nucleic acid content. A molecular weight of 4.6 × 10⁶ was calculated from the sedimentation coefficient, S°_20,w = 86.2S, the diffusion coefficient, D_20,w = 1.55 × 10^-7 cm²/sec., and an assumed partial specific volume, [UNK] = 0.708 ml/gm. The virus has a ribonucleic acid content of 1.0 × 10⁶ atomic mass units. Electrophoresis experiments showed that the virus is stable in 0.10 ionic strength buffers in the pH range 3-6. Breakdown of the virus was observed outside this pH range. Some characteristics of the breakdown products are described.     

Ion exchange chromatography of proteins-prediction of elution curves and operating conditions. I. Theoretical considerations

A mathematical model is proposed for the elution of proteins on ion exchange columns by a linear gradient increase and stepwise increase of ionic strength in order to predict relationships between the elution characteristics (the peak position, the peak width, etc.) and the operating conditions (the flow rate, the slope of gradient, etc). This model is in principle based on the continuous-flow plate theory, in which the protein concentration and ionic strength dependent distibution coefficient between proteins and ion exchangers and zone sperading effects are taken into consideration. The advantage of this model is its simplicity since it requires only two parameters: The distribution coefficient and the number of plates. Since the distribution coefficient of proteins depends on both the protein concentration and ionic strength of the elution buffer, the number of plates should vary with time. However, it is extremely difficult to take into consideration the time-dependent number of plates. Therefore, we assume that the number of plates is constant and related to that number derived from a mass balance model which includes longitudinal dispersion and gel phase diffusion. On the basis of these assumptions, a method for determining the number of plates by the moment method is presented. Although the dependencies of the peak position and peak width on the slope of linear gradient are predictable by numerical calculations of the present model, simpler methods for prediction of these dependencies are desirable. A graphical method is proposed for prediction of the peak position. For prediction of the peak width, an asymptotic solution is derived from a quasi-steady-state model.     

Molecular weight estimation using sodium dodecyl sulfate-pore gradient electrophoresis

Pore gradient electrophoresis (PGE) in the presence of sodium dodecyl sulfate (SDS) provides a means for high resolution fractionation of multicomponent protein systems and permits estimation of molecular weights for macromolecules ranging from 10³ to 10⁶. We have evaluated the performance of several methods used to construct calibration curves for estimation of molecular weights using SDS-PGE. A linear relationship between the logarithm of molecular weight, log (M_r), and the logarithm of the relative mobility, log (R_l), can be obtained for a 30-fold range of molecular weights. However, this range of linearity depends on the choice of the concentration gradient, the degree of crosslinking of the gel, and on the nature of the underlying relationship between the retardation coefficient, K_R, and the molecular weight. An empirical relationship, first introduced by Lambin et al. (1976, Anal. Biochem.74, 567) between log (M_r) and the logarithm of the gel concentration at the position reached by the protein, log (%T), provides better linearity over a wider molecular weight range than does the use of log (R_l). We have compared these relatienships by experimental analysis of 10 standard proteins and by a theoretical analysis of an idealized model system. A computer program has been developed which provides appropriate statistical estimation of the molecular weight for an unknown protein, together with its standard error and 95% confidence limits. A new method has also been developed for analysis of nonlinear calibration curves in terms of molecular weight versus distance migrated, based on a theoretically justifiable, physical-chemical model. This model implies that either the relationship between log (M_r) and log (R_l) or the one between log (M_r) and log (%T) will become nonlinear as the range of molecular weight is extended. We suggest that the use of a nonlinear least-squares curve-fitting procedure provides an optimal method for molecular weight estimation when sufficient data are available. Based on these findings, a general strategy is presented for estimation of molecular weights by polyacrylamide gel electrophoresis.     

Thresholds in field-induced reactions of linear biopolymers: Strong chain-length dependence of field effects in DNA

We have analysed the field-induced conformation change of DNA by absorbance measurements at the magic angle. Conformation changes are observed when the electric field strength exceeds a clearly defined threshold value. The threshold values increase with increasing salt concentration and show a linear dependence upon the logarithm of the ionic strength. Measurements with homogeneous DNA samples of different chain lengths N show that the threshold increases with decreasing N; at a given ionic strength the threshold is a linear function of the logarithm of N. The threshold value observed for a circular DNA molecule with a chain length N_c fits to these data with an effective length N_c/2. This result indicates that the length of maximal extension is important for the field-induced reaction and suggests, together with the other results, that the field-induced reaction is mainly driven by a polarization of the ion atmosphere along the axis of DNA, Some data are also given for the dynamics of the reaction: at high electric field pulses the first step is a fast deslacking and lilting of the bases followed by a slow unwinding process. For short pulses the reaction is almost completely reversible with a characteristic time constant of about 3 μs for the back reaction.     

Comparison of molecular weight determination of sodium alginate by sedimentation-diffusion and light scattering

The weight average molecular weight, M̄_w, of sodium alginates were determined by the sedimentation-diffusion technique using photon correlation spectroscopy rather than boundary spreading in the analytical ultracentrifuge to determine the translational diffusion coefficients. This enables the diffusion coefficients to be determined easily and accurately. Excellent correlation is found between the observed zero concentration translational diffusion coefficient, D_O and the M̄_W values in a emphirical power law. The M̄_W obtained by sedimentation-diffusion and laser light scattering compare very favourably. The concentration dependence of the photon correlation spectroscopy data allowed determination of the coil overlap concentration, c*. The inverse proportionality of c* to both M̄_W and [η] is demonstrated.     

Disc electrophoretic studies of hemoglobin dissociation by p-hydroxymercury benzoate

The reaction of excess p-mercurybenzoate with human hemoglobin produces five electrophoretic species on polyacrylamide gel. Only two of these bands have been previously observed when starch gel was employed. The chemical and electrophoretic studies presented in this paper illustrate that the appearance of an “extra” band in the β zone is due to the ability of PAGE to separate the βb₂^PMB ? β^PMB equilibrium to its discrete components. The remaining bands are accounted for by the superior resolution of PAGE over starch gel electrophoresis which allowed the detection of two (αβ)^PMB dimer species.