Density-gradient sedimentation equilibrium of DNA and the effective density gradient of several salts

The various treatments of sedimentation equilibrium are compared on a theoretical and an experimental basis. Particular attention is paid to the polyelectrolyte nature of the problem and the choice of a neutral component. The effective density gradients of several cesium salts for DNA are measured. Two previous theories for the effective density gradient are shown to be equivalent, and the experimental values are interpreted with respect to these theories. It is clear t hat sedimentation equilibrium in a density gradient may be used for the determination of unambiguous molecular weights.     

The flexibility of low molecular weight double-stranded dna as a function of length: I. Isolation and physical characterization of seven fractions

Sonicated calf thymus DNA was fractionated by rate zonal centrifugation into seven fractions with weight average molecular weights ranging from 0.28 to 1.3 × 10⁶ daltons, as determined by sedimentation equilibrium and light scattering measurements (the latter are described in the accompanying paper). Electron microscopy and sedimentation equilibrium analysis revealed these fractions to be narrowly disperse with M_w/M_n ratios averaging about 1.06. Intrinsic viscosities and sedimentation rates were measured and found to vary linearly with molecular weight in double-logarithmic plots in fair agreement with previously published functions relating these parameters for low molecular weight DNA. The average value for β from the Mandelkern— Flory equation was 2.59 × l0⁶, also agreeing with reported estimates of this parameter for short DNA. These data will be used in the second paper of this series to calculate the persistence length of the DNA fragments in each of the seven fractions by light scattering and hydrodynamic theories for the Kratky—Porod worm-like coil.     

Sizes and mass distributions of clathrin-coated vesicles from bovine brain

Clathrin-coated vesicles obtained from bovine brain have been studied by ultracentrifugation and dynamic light scattering techniques to provide information on their sedimentation and mass distributions and their average diffusion coefficients. "Uncoated" vesicles, obtained by removing the protein coat from coated vesicles, have been similarly characterized. For typical preparations, maximal values of approximately 210 and 95 S are observed for the sedimentation coefficients of coated and uncoated vesicles, respectively. Corresponding values for the average molecular weights, determined from values of average sedimentation and diffusion coefficients, are 49 X 10(6) and 13 X 10(6); values obtained by equilibrium sedimentation are 37.2 X 10(6) and 10.6 X 10(6). In order to obtain these results, some minor modifications of sedimentation and light-scattering techniques have been devised which may have application to other studies of size distributions of large particles.     

Molecular weight determination of membrane proteins by sedimentation equilibrium at the sucrose or nycodenz-adjusted density of the hydrated detergent micelle

The determination of the molecular weight of a membrane protein by sedimentation equilibrium is complicated by the fact that these proteins interact with detergents and form complexes of unknown density. These effects become marginal when running sedimentation equilibrium at gravitational transparency, i.e., at the density corresponding to that of the hydrated detergent micelles. Dodecyl-maltoside and octyl-glucoside are commonly used for dissolving membrane proteins. The density of micelles thereof was measured in sucrose or Nycodenz. Both proved to be about 50% lower than those of the corresponding non-hydrated micelles. Several membrane proteins were centrifuged at sedimentation equilibrium in sucrose- and in Nycodenz-enriched solutions of various densities. Their molecular weights were then calculated by using the resulting slope value at the density of the hydrated detergent micelles, i.e. at gravitational transparency, and the partial specific volume corrected for a 50% hydration of the membrane protein. The molecular weights of all measured membrane proteins, i.e. of photosystem II complex, reaction center of Rhodobacter sphaeroides R26, spinach photosystem II reaction center (core complex), bacteriorhodopsin, OmpF-porin and rhodopsin from Bovine retina corresponded within +/-15% to those reported previously, indicating a general applicability of this approach.     

Sensitivity of gelatin and albumin to irradiation at 230 NM.

Gelatin or poly-L-tyrosyl gelatin shows extensive degradation when exposed to ultraviolet radiation at a wavelength of 230 nm. Bovine serum albumin, a globular protein, exposed either to such ultraviolet radiation or to gamma-irradiation in the solid state resists the photolysis of peptide bonds. Molecular weights are determined by the ultracentrifugal "low speed" sedimentation equilibrium method. The effect of different speeds on the apparent average molecular weight of heterogenous material is clearly illustrated.     

Physical and chemical properties of the NH2-terminal glutamic acid and lysine forms of human plasminogen and their derived plasmins with an NH2-terminal lysine heavy (A) chain.

Comparative physical and chemical data are described for the human NH2-terminal Glu-plasminogen and Lys-plasminogen forms in order to determine the exact relationship between these two types of the zymogen. The molecular weights of Glu-plasminogen and Lys-plasminogen were similar and were determined to be 83, 800 plus or minus 4, 500 and 82, 400 plus or minus 3, 300, respectively, by sedimentation equilibrium methods. The molecular weights were identical in dodecyl sulfate solutions, approximately 83, 000, by sedimentation equilibrium methods. The sedimentation coefficients, s-020, w of Glu-plasminogen and Lys-plasminogen were determined to be 5.0 S, and 4.4 S, respectively. These two plasminogen forms had different partial specific volumes, and calculations of the frictional coefficients from sedimentation coefficients and molecular weights indicated conformation differences. Glu-plasminogen appeared to be larger in size than Lys-plasminogen in acrylamide gel-dodecyl sulfate electrophoresis. The amino acid compositions of Glu-plasminogen and Lys-plasminogen, and their major isolated isoelectric forms, were found to be similar, but several amino acid residues (glutamic acid, alanine, isoleucine, phenylalanine, and lysine) were found to be significantly higher in the Glu-plasminogen forms. The derived plasmins from both the Glu- and Lys-plasminogens with an nh2-terminal Lys- heavy (A) chain were found to have identical molecular weights of 76, 500 plus or minus 2, 500, and sedimentation coefficients, s-020, w of 4.3 S.     

Studies on regulatory functions of malic enzymes. VI. Purification and molecular properties of NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme [EC 1.1.1.40] was highly purified from Escherichia coli W cells. The purified enzyme was homogeneous as judged by ultracentrifugation and gel electrophoresis. The apparent molecular weights obtained by sedimentation equilibrium analysis, from diffusion and sedimentation constants, and by disc electrophoresis at various gel concentrations were 471,000, 438,000, and 495,000, respectively. The subunit molecular weights obtained by sedimentation equilibrium analysis in the presence of 6 M guanidine hydrochloride and gel electrophoresis in the presence of sodium dodecyl sulfate were 76,000 and 82,000, respectively. The sedimentation coefficient (S(0)20, W) was 13.8S, and the molecular activity was 44,700 min-1 at 30 degrees C. The amino acid composition of the enzyme was determined, and the results were compared with those of NAD-linked malic enzyme from the same organism and those of pigeon liver NADP-linked malic enzyme. The partial specific volume was calculated to be 0.738 ml/g. The Km value for L-malate was 2.3 mM at pH 7.4. Malonate, tartronate, glutarate, and DL-tartrate competitively inhibited the activity. The saturation profile for L-malate exhibited a marked cooperativity in the presence of both chloride ions and acetyl-CoA. However, acetyl-CoA alone did not show cooperativity or produce inhibition in the absence of chloride ions. Vmax and Km were determined as a function of pH. The optimum pH for the reaction was 7.8. Inspection of the Dixon plots suggested that three ionizable groups of the enzyme are essential for the enzyme activity. In addition to the oxidative decarboxylase activity, the enzyme preparation exhibited divalent metal ion-dependent oxaloacetate decarboxylase and alpha-keto acid reductase activities. Based on the above results, the molecular properties of the enzymatic reaction are discussed.     

Sedimentation-equilibrium studies on the heterogeneity of two β-lactamases

Solutions of crystalline beta-lactamase I and beta-lactamase II, prepared by Kuwabara (1970), were examined in the ultracentrifuge and their sedimentation coefficients, diffusion coefficients, molecular weights and heterogeneity determined. Each sample was shown to consist of a major component comprising at least 97% of the material and a minor component of much higher molecular weight. The molecular weights of the major components were 27800 for beta-lactamase I and 35600 for beta-lactamase II. Emphasis is placed on a straightforward practical way of analysing the sedimentation-equilibrium results on mixtures of two macromolecular components rather than on a strict theoretical solution. Appendices describe the theory of systems at both chemical and sedimentation equilibrium and the procedure for calculating the combined distribution of two components.     

Characterization of the terminal degradation products of canine fibrinogen by plasmin.

Fibrinogen, isolated from canine plasma by the successive procedures of (1) freezing and thawing, (2) fractional precipitation with 25% saturated (HN4)2SO4 and (3) Sepharose 6B gel-filtration, had a molecular weight of 282 000 by the rapid sedimentation equilibrium method. However, a molecular weight for canine fibrinogen of 332 000, which is closer to that reported for human and bovine fibrinogens (340 000 plus or minus 20 000), was obtained from the sum of the molecular weights of the Aalpha, Bbeta and gamma chains, determined from dodecylsulfate gel electrophoretic patterns of reduced fibrinogen. Canine fibrinogen, subjected to proteolysis by urokinase-activated plasminogen for 24 h, contained degradation fragments D and E which were isolated by starch block electrophoresis and Sephadex G-200 gel-filtration. The purified D and E fragments with sedimentation coefficients of 5.0 S and 2.5 S had weight average molecular weights of 89 000 and 42 000, respectively by the rapid sedimentation equilibrium method. The ratio of D to E was 2:1 per parent fibrinogen molecule. Antigenic analysis according to anti-fibrinogen antiserum showed that both D and E fragments were antigenically deficient to native fibrinogen and revealed a reaction of non-identity with each other. Upon immunoelectrophoresis at pH 8.2, D and E had different electrophoretic mobilities. Preliminary studies indicate that based on thrombin time alone, D has anticoagulant activity while E appears to be a coagulation potentiator. Canine fibrinogen apparently consist of two core fragments with dissimilar chemical characteristics in common with the fundamental structures of human and bovine fibrinogens.     

SED88: a Pascal program for the analysis of sedimentation equilibrium data

Analytical ultracentrifugation is commonly used for the determinationof molecular weights (sedimentation equilibrium) and sedimentationcoefficients (sedimentation rate) of biological macromoleculesin solution. A Turbo Pascal program for the analysis of sedimentationequilibrium centrifugation data produced by absorbance opticalsystems is described. The user may enter data from a scan ofabsorbance versus distance from the centre of rotation, viaa graphics tablet (or ASCII file). This is subsequently manipulatedto yield an apparent weight average molecular weight for thegiven sample. Plots of In (absorbance) versus (radius²) mayalso be produced. The method described uses readily availablecomputational equipment requiring only a graphics tablet inaddition to an IBM PC compatible computer. This technique andthe software developed have been used to investigate the molecularweight range of two International Humic Substances Society (IHSS)reference samples from the Suwannee River. Received on October 7, 1988; accepted on December 12, 1988     

Salt-dependent interconversion of inner histone oligomers.

The inner histone complex, extracted from chicken erythrocyte chromatin in 2 M NaCL AT pH 7.4, has been characterized by sedimentation equilibrium and sedimentation velocity. High speed sedimentation equilibrium studies indicate that in 2 M NaCl the inner histones are a weakly associating system with contributions from species ranging in molecular weight from dimer to octamer. The appearance of a single boundary (3.8S at 2 M NaCl) in sedimentation velocity studies conducted over a wide range of protein concentrations and ionic conditions indicates that the various histone oligomers present are in rapid equilibrium with one another. At higher salts the equilibrium is shifted to favor higher molecular weight species; in 4 M NaCl essentially all of the histone is octameric at protein concentrations above 0.2 mg/ml. The facile interconversion of histone oligomers suggests that small alterations in histone-histone interactions may be responsible for changes in nucleosome conformations during various biological processes.     

Sedimentation equilibrium in macromolecular solutions of arbitrary concentration. II. Two protein components

Relations describing sedimentation equilibrium in solutions containing two macromolecular solute components are derived for the following cases: (1) two nonassociating proteins at arbitrary concentration, (2) one dilute self-associating protein in the presence of a second inert protein at arbitrary concentration, and (3) two proteins at arbitrary concentration that can associate to form a single heterocomplex of arbitrary composition. As in earlier work (R. C. Chatelier and A. P. Minton (1987) Biopolymers, 26, 507–524), the relations are obtained by using scaled particle theory to calculate the thermodynamic activity of each species present at a given radial distance in the centrifuge. The results of numerical simulations of sedimentation equilibrium are presented as the dependence of apparent molecular weights, or apparent weight-average molecular weights, upon solution composition. Semiempirical methods are presented, by means of which the weight-average molecular weights of self- and heteroassociating proteins in highly nonideal solutions may be estimated from experimental data. It is found that the semiempirical methods yield reasonably accurate estimates of the true weight-average molecular weight over a broad range of experimental conditions, providing that the partial specific volumes of two components in a heteroassociating system do not differ by more than about 0.05 mL/g.     

Physico-chemical studies on di-iodotyrosine dextran

Dextran is currently being considered as a carrier biopolymer system for drug targeting. Di-iodotyrosine (DIT), and its radioactive derivative are useful markers for identifying the in-vivo route of these substrates. The performance of these substances in vivo is closely linked to their physico-chemical properties in solution. These properties have been investigated on a series of gel permeation chromatography fractionated DIT-dextrans using a combination of sedimentation, viscometric and laser-light scattering techniques. Weight average molecular weights, M_w, from sedimentation equilibrium and light scattering range from 0·0195 to 0·054 × 10⁶. Double logarithmic representations of M_w versus sedimentation coefficient, s^o_20.w, and intrinsic viscosity [η] suggest that these labelled dextrans have a more compact conformation in solution than do their unlabelled analogues — which behave as random coils. The implications of this for drug delivery are indicated.     

Molecular Weight Determination of Gliadin Fractions in Gel Filtration by SDS-Polyacrylamide Gel Electrophoresis and Sedimentation Equilibrium

Gliadin was fractionated into three fractions; ω-gliadin, Fraction III (γ-gliadin) and Fraction IV (α- and β-gliadin). The determination of the molecular weights (MW) of the three fractions was performed by both SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and sedimentation equilibrium. In SDS–PAGE, ω-gliadin gave three bands (MW 50,000, 54,000 and 64,000), Fraction III two bands (MW 38,000 and 46,000) and Fraction IV two bands (MW 33,000 and 38,000), The sedimentation analysis showed that each fraction was fairly homogeneous relative to molecular weight. The molecular weights obtained by sedimentation were 28,000 for Fraction III and 27,000 for both Fraction IV and ω-gliadin. The disagreement in molecular weight between sedimentation and gel electrophoresis was discussed.     

Caldesmon. Molecular weight and subunit composition by analytical ultracentrifugation

A wide range of values has been reported for the subunit and molecular weights of smooth muscle caldesmon. There have also been conflicting reports concerning whether caldesmon is a monomer or dimer. We attempted to resolve these uncertainties by determining the molecular weight of chicken gizzard smooth muscle caldesmon using the technique of sedimentation equilibrium in the analytical ultracentrifuge. Unlike previous methods that have been used to estimate the molecular weight of caldesmon, the molecular weight determined by equilibrium sedimentation does not depend upon assumptions about the shape of the molecule. We concluded that caldesmon in solution is monomeric with a molecular mass of 93 +/- 4 kDa, a value that is much less than those previously reported in the literature. This new value, in conjunction with sedimentation velocity experiments, led to the conclusion that caldesmon is a highly asymmetric molecule with an apparent length of 740 A in solution. The mass of a cyanogen bromide fragment, with an apparent mass of 37 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was determined to be 25.1 +/- 0.6 kDa using sedimentation equilibrium. These results imply that the reported molecular weights of other fragment(s) of caldesmon have also been overestimated. We have determined an optical extinction coefficient for caldesmon (E1%(280 nm) = 3.3) by determining its concentration from its refractive index which was measured in the analytical ultracentrifuge. From the above values of the molecular weight and the extinction coefficient, we redetermined that the caldesmon molecule has two cysteines and recalculated the stoichiometric molar ratio of actin/tropomyosin/caldesmon in the smooth muscle thin filament to be 28:4:1.     

Properties of Two Homologous Alkaline Proteases from Streptomyces rectus

Some physicochemical properties of two thermostable proteases from Streptomyces rectus are described. The enzymes were judged to be identical with respect to molecular weight, inactivation with serine protease inhibitors, and in primary structure by peptide analysis. Amino acid analysis indicated the enzymes had identical compositions except for their amide content. The molecular weights of the enzymes were judged to be 28,000 by sedimentation equilibrium, 26,200 by sedimentation diffusion, and 29,100 from amino acid analysis. Titration of the proteases with diisopropylfluorophosphate and phenylmethane sulfonylfuoride indicate equivalent weights of 28,500 and 32,800 g, respectively, for the proteins. The pentapeptide around the serine residue reacting with diisopropylfluorophosphate was isolated and had the composition: Asx(1), Gly(1), Thr(1), Ser(1), Met(1).     

Size and shape of two intestinal dipeptidases

Physicochemical parameters were determined on glycyl-L-leucine hydrolase (glycy-leucine dipeptidase, EC 3.4.13.2) and aminoacyl-L-proline hydrolase (proline dipeptidase, EC 3.4.13.9), purified from pig small intestine. The native molecular weights were found to be 115,000 and 113,000, respectively, as determined by a sedimentation equilibrium technique. Under denaturing conditions the molecular weights were found to be 51,000 and 63,200, respectively, using the same technique. It is concluded that each dipeptidase is composed of two subunits of equal molecular weight. The two dipeptidases have the same Stokes radius, 4.2 nm, analysed by gel chromatography. The sedimentation coefficients were found to be 5.8. S and 6.5 S and the intrinsic viscosities 5.4 ml/g and 5.8 ml/g, respectively. For both dipeptidases the measured physicochemical parameters are in accordance with the model of a prolate ellipsoid of revolution, having an axial ratio of about 5.     

The molecular weight of Ehrlich tumor cell ribonucleotide reductase and its subunits: Effector-induced changes

The molecular weights of Ehrlich tumor cell ribonucleotide reductase and its individual components were determined by sedimentation equilibrium in the Beckman Airfuge. The distribution of enzyme after sedimentation equilibrium was determined by measurement of the CDP reductase and ADP reductase activities associated with ribonucleotide reductase. The apparent molecular weight of the intact enzyme was 304,000 when assayed for CDP reductase and 254,000 when assayed for ADP reductase. This difference in apparent molecular weights was statistically significant with a P value of 0.0002. The molecular weights of the individual components of ribonucleotide reductase were determined in a similar fashion by assaying in the presence of an excess of the complementary component. The non-heme iron component had a molecular weight of 81,000 when assayed for either CDP or ADP reductase activity. The effector-binding component had an apparent molecular weight of 127,000 when assayed for CDP reductase and 95,000 when assayed for ADP reductase. This difference in apparent molecular weights was statistically significant with a P value of 0.004. The effectors ATP and dGTP altered the apparent molecular weights of the intact enzyme and individual components. In the presence of ATP the molecular weight of intact CDP reductase was 481,000 while the apparent molecular weight of the effector-binding component of CDP reductase alone was 418,000. In the presence of dGTP, the molecular weight of intact ADP reductase was 293,000 while the apparent molecular weight of the effector-binding component of ADP reductase alone was 154,000. These results indicate that the proportion of the non-heme iron component and the effector-binding component is not equimolar and that the composition of the enzyme is not constant but is altered by the presence of effectors. Our data also suggest that CDP reduction and ADP reduction are catalyzed by different molecular species of the enzyme which apparently have different effector-binding components.     

A comparison of molecular mass determination of hyaluronic acid using SEC/MALLS and sedimentation equilibrium

Hyaluronic acid (HA) is a natural polysaccharide with importance in the pharmaceutical, medical and cosmetic industries. Determining factors in its final applications are its physicochemical properties, particularly molecular mass. A high molecular mass HA was degraded using five different hydroxyl free-radical starting concentrations chemically produced from ascorbic acid and hydrogen peroxide. The aims of the study were to investigate the effect of different hydroxyl free-radical concentrations on the chain length of HA and compare the molecular masses obtained from analytical ultracentrifugation using sedimentation equilibrium experiments and size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). The results indicated that their molecular masses varied, depending on the degree of hydroxyl free-radical starting concentration. Molecular mass values obtained from sedimentation equilibrium experiments for each sample showed the same trend as those obtained from the SEC/MALLS in the range of molecular masses studied. The molecular masses obtained from sedimentation equilibrium for high molecular mass samples from reciprocal plots of apparent weight average molecular mass against concentration gave values similar to those obtained by SEC/MALLS. In contrast, the molecular mass from conventional plots for high molecular mass samples were much lower than those from SEC/MALLS, even when high ionic strength buffers were used.     

Anomalous molecular weights to proteases in gel chromatography

Attention is drawn to the fact that, in comparison to those values derived from sedimentation equilibrium experiments, gel chromatographic methods give anomalously low molecular weights for a variety of proteases. This appears to be particularly true for extracellular proteases of broad specificity produced by bacteria and fungi.