Correct Molecular Weight Research Articles

Light Scattering and Viscometric Study of Cellulose in Aqueous Lithium Hydroxide

An attempt was made to determine molecular parameters of cellulose dissolved in simple solvents. Cellulose regenerated from a cuprammonium solution of acid-hydrolized cotton linters was found to dissolve molecularly, without suffering molecular degradation, into 6 wt% aqueous LiOH solution by choosing an adequate condition. The apparent weight-average molecular weight Mw*, of cellulose obtained by light scattering in a non-dialyzed aqueous LiOH, was converted into the correct weight-average molecular weight Mw with the aid of the data on Mw* and Mw for a dialyzed cadoxen solution of cellulose. The Mark–Houwink–Sakurada equation for cellulose in aq LiOH was [η]=2.78×10−2 Mw0.79 at 25°C {[η]; limiting viscosity number}. The Flory’s viscosity parameter Φ for this system was significantly smaller than the theoretical value for unperturbed chains at non-draining limit, showing a molecular weight dependence. The linear expansion factor for the radius of gyration was always below 1.04 as estimated from the penetration function. The unperturbed chain dimensions were estimated by various methods using the light scattering and/or viscometric data. Cellulose was found to be semiflexible in aq LiOH.

Dependence of chain conformation and solution thermodynamics on composition for poly(ortho ester)s prepared from ketene acetals and mixtures of diols

AbstractThe solution properties of a series of random copolymers prepared from ketene acetals with mixtures of diols have been characterized by dilute solution light scattering, chromatographic, and viscometric techniques. Five solvents with refractive indices ranging from 1.4 to 1.5 were used to obtain the correct weight average molecular weight. Changes in the unperturbed radius of gyration, obtained by combined size exclusion chromatography and low‐angle light scattering techniques, parallelled that predicted by simple considerations of changes in repeat unit size. Thus the relative contributions of differing copolymer–solvent interactions and changing unperturbed radii of gyration to the hydrodynamic volume of random copolymers of different compositions were evaluated.

Immunoextraction of lipoamide dehydrogenase from cultured skin fibroblasts in patients with combined α-ketoacid dehydrogenase deficiency

Combined deficiency of the pyruvate, α-ketoglutarate and branched-chain keto acid dehydrogenase complexes is a rare condition in which activity of lipoamide dehydrogenase is either reduced or grossly deficient. Activities in three cell strains from patients with excretion of branched chain ketoacids and α-ketoglutarate and lactic-acidemia showed decreased levels of the three α-ketoacid dehydrogenases. Lipoamide dehydrogenase activity was 5% of normal in one cell stain and 50–60% in the other two. Antiserum raised against lipoamide dehydrogenase was used to immunoprecipitate labelled lipoamide dehydrogenase from fibroblasts grown on [ 35S]methionine. After separation of cell proteins from control fibroblasts by sodium dodecyl sulphate polyacrylamide gel electrophoresis and fluorography, a prominent 55 kilodalton band was evident in cell extracts treated with the antiserum which corresponded to lipoamide dehydrogenase. In the cell lines from patients with combined α-ketoacid dehydrogenase deficiency immunoprecipiation of lipoamide dehydrogenase showed that this protein was present in similar amounts to that seen in control cell lines and was also of the correct molecular weight.

Molecular Cloning and In Vitro Expression of a cDNA Clone for Human Cellular Tumor Antigen p53

Three clones for the human tumor antigen p53 were isolated from a cDNA library prepared from A431 cells. One of these clones, pR4-2, contains the entire coding region for human p53. This clone directs the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of an authentic p53 molecule in an in vitro transcription-translation reaction. Although the pR4-2 clone contains the coding region for p53, it is not a full-length copy of the human p53 mRNA. Northern analysis showed that the p53 mRNA is approximately 2,500 nucleotides long, whereas the pR4-2 insert is only 1,760 base pairs in length. Analysis of the DNA sequence of this clone suggests that the human p53 polypeptide has 393 amino acids. We compared the predicted amino acid sequence of the pR4-2 clone with similar clones for the mouse p53 and found long regions of amino acid homology between these two molecules.

Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen p53.

Three clones for the human tumor antigen p53 were isolated from a cDNA library prepared from A431 cells. One of these clones, pR4-2, contains the entire coding region for human p53. This clone directs the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of an authentic p53 molecule in an in vitro transcription-translation reaction. Although the pR4-2 clone contains the coding region for p53, it is not a full-length copy of the human p53 mRNA. Northern analysis showed that the p53 mRNA is approximately 2,500 nucleotides long, whereas the pR4-2 insert is only 1,760 base pairs in length. Analysis of the DNA sequence of this clone suggests that the human p53 polypeptide has 393 amino acids. We compared the predicted amino acid sequence of the pR4-2 clone with similar clones for the mouse p53 and found long regions of amino acid homology between these two molecules.

Transformation of B and non‐B cell lines with the 2,4,6‐ trinitrophenyl (TNP)‐specific immunoglobulin genes

The rearranged mu and kappa genes from the 2,4,6-trinitrophenyl (TNP)-specific hybridoma Sp6 have been introduced into B cells from three different stages of differentiation as well as 5 non-B cell lines to determine the levels and modes of immunoglobulin (Ig) gene expression. In pre-B cells transformed with the mu and kappa genes, low levels of Sp6-specific mu RNA were produced and approximately 210-fold less mu and 800-fold less kappa proteins were produced than in the hybridoma Sp6. The Ig proteins were present intracellularly, but were not detected on the cell membrane. In mature surface sIg+ B cell transformants, higher levels of mu Sp6 and kappa Sp6 proteins and RNA were produced than in the pre-B cell transformants (12 X mu, 70 X kappa). These transformants displayed the mu Sp6 and kappa Sp6 proteins on the cell membrane and also secreted the transfected Ig product. Plasma cell transformants produced the highest amounts of mu Sp6 and kappa Sp6 proteins. These transformants secreted pentameric IgM but did not display detectable amounts of these proteins on the cell membrane. T cell and one fibroblast transformant produced Ig as normal sized mu Sp6 and kappa Sp6 proteins. All other mu Sp6 and kappa Sp6 non-B cell transformants (melanoma, teratoma and macrophage) failed to produce enough Ig to determine whether the Ig proteins were of the correct molecular weights. The T cell and fibroblast transformants that produced Ig proteins did not secrete or display detectable Ig on the cell membrane. The expression of Ig did not inhibit the expression of the T cell antigen Thy-1 in the T cell transformants.

Inhibition of acrosin by oviductal secretory immunoglobulin A

AbstractA major inhibitor of acrosin in rhesus monkey and rabbit oviduct fluid, isolated by isoelectrofocusing in sucrose gradients, displayed a broad peak in the acidic region of the column and was demonstrated to contain secretory IgA specific for acrosin. Its identity was established by immunodiffusion, by the removal of acrosin inhibition with antisera to IgA (α‐chain), and by its correct molecular weight during ultracentrifugation. Purified human serum IgA also inhibited rabbit, rhesus monkey, and human acrosins, but neither purified human IgG nor IgM had any inhibitory effect on these acrosins. Neither oviduct fluid secretory IgA nor purified human serum IgA inhibited the activity of bovine pancreatic trypsin. The high specificity of secretory IgA for acrosin and its presence in every rabbit and rhesus monkey oviduct fluid specimen examined suggests a possible regulatory role for this antibody in reproduction.

Analysis of the yolk proteins in Drosophila melanogaster: Translation in a cell free system and peptide analysis

It has been established that the yolk proteins in Drosophila are synthesised and secreted by the fat body, transported in the haemolymph, and accumulated in the developing oocytes [l-5] . In this paper we describe experiments which show that YPl, and YP2 are translated in a cell-free system as proteins of the correct molecular weight rather than aslarger precisors, furthermore our data indicates that the smallest yolk protein, YP3, may be a post-translationally modified derivative of YP2.

Compressibility studies of three proteins in CsCI solutions in the analytical ultracentrifuge

AbstractThe compositional buoyant densities, ρ;, of human γ‐immunoglobulin, bovine serum mercaptalbumin, and egg albumin have been measured in CsCl solutions in the analytical ultracentrifuge as a function or pressure. Standard pressure coefficients, ψ0, and standard partial specific volumes of the solvated proteins, υ,0, have been computed from these data. The ψ0 values obtained are strikingly different from each other and from the only other pressure coefficients which have been measured, those values obtained for nucleic acids and nucleoproteins. The ψ value for γ‐immunoglobulin is negative, the first nonpositive value obtained, and suggests an unusual internal structure for this protein. The pressure coefficient of mercaptalbumin is not constant. A second‐order relation is derived and utilized to interpret these data. The slope of the ρ(P) plot for egg albumin was constant and negative and yielded values of ψ0 which are about 20% as large as those reported for DNA. Evaluation of published isopiestic data for egg albumin in CsCl solutions provided the dependence of preferential hydration on water activity. This quantity, (dΓ′/da) as well as α, were found to be negative. The values of ψ0 and α were used to compute the effective density gradient from which the correct molecular weight of egg albumin was obtained. The apparent specific volume of egg albumin in a buoyant CsCl solution was measured using the Mettler‐Paar densimeter.

Localization of eukaryotic initiation factor 3 on native small ribosomal subunits

The localization of eukaryotic initiation factor 3(eIF-3) on native small ribosomal subunits has been established by electron microscopy through a comparison of native small ribosomal subunits with derived subunits and with native subunits stripped of eIF-3. Small subunits derived from reticulocyte ribosomes by the puromycin/KCl method are seen in electron micrographs as elongated particles, divided by a heavily stained partition into approximately one-third and two-third domains. Most particles (60-70%) observed in electron micrographs of native small subunit preparations resemble derived small subunits, but have an additional mass attached to one side, thus producing profiles with a three-lobed appearance. The mass measures approximately 160 x 100 x 60 A, and its particle weight is estimated to be about one-third to one-half that of a 40S subunit. The site of attachment of the additional mass is located on a prominence extending from the central part of the small subunit and is separated by a cleft from the smaller third of the subunit. The remaining particles in preparations of native subunits resemble the profiles seen in electron micrographs of derived subunits. After removal of eIF-3 by treatment with high concentrations of salt, profiles observed in electron micrographs of washed, native subunits were indistinguishable from those of derived subunits. Since removal of eIF-3 coincided with removal of a mass of the correct molecular weight, subunits with the three-lobed appearance are identified as native small subunits carrying eIF-3.

Small-angle x-ray scattering study of S4-RNA, the 16 S RNA binding site for the protein S4 from Escherichia coli ribosomes

The ribonucleic acid (16 S RNA) of the 30 S Escherichia coli ribosomal subunit binds many of the proteins in this subunit, such as, for instance, S4, S7, S8, S15 and S20 [1,2]. The 16 S RNA binding site for the S4 protein, S4-RNA, can be isolated in pure form [3-91 ; and it is indicated that this binding site consists mainly of two regions of approximately 150 and 160 nucleotides, originating from the 5’ terminal third of the 16 S RNA molecule [7]. These two regions, which are separated by about 120 nucleotides of the 16 S RNA sequence, seem to be stabilized by a specific RNA-RNA interaction [7] . Although the sequence of the S4-RNA region, prepared with both T1 and pancreatic ribonucleases, has been partially analysed [7,9], the correct molecular weight and the size and shape of S4-RNA are not known. We have analysed S4-RNA by using the small-angle X-ray scattering method; the results yield a molecular weight of 136 000 and a radius of gyration of 43.5 A. The X-ray scattering curve can be explained, in its proximal angular range, by the scattering from a twoparameter, uniform electron density model with a shape of an oblate ellipsoid and the dimensions of 132 X 132 X 32 a. 2. Materials and methods

Increased membrane binding of erythrocyte catalase in hereditary spherocytosis and in metabolically stressed normal cells

Normal red blood cell (RBC) membranes were compared with (1) RBC membranes from six patients with hereditary spherocytosis (HS), (2) normal membranes after hemolysis of the RBC in the presence of calcium, or (3) normal membranes after incubation of RBC for 24 hr in phosphate-buffered saline containing calcium without added glucose. When compared with normal controls, polyacrylamide gel electrophoresis with sodium dodecyl sulfate (PAGE SDS) of all three preparations showed an increase in membrane binding of globin and protein band 4.5 (60,000 molecular weight). In an attempt to identify band 4.5, 14 enzymes were assayed in the RBC membranes. Of these, catalase and lactate dehydrogenase were increased in membranes from HS RBC and from normal cells exposed to calcium. Only catalase, however, was present in sufficient quantity and had the correct subunit molecular weight on PAGE SDS and calcium-dependent membrane binding to account for an appreciable portion of 4.5. Caralase was further identified with a component of band 4.5 by double immunodiffusion using a specific anti-catalase antibody.

Increased Membrane Binding of Erythrocyte Catalase in Hereditary Spherocytosis and in Metabolically Stressed Normal Cells

Abstract Normal red blood cell (RBC) membranes were compared with (1) RBC membranes from six patients with hereditary spherocytosis (HS), (2) normal membranes after hemolysis of the RBC in the presence of calcium, or (3) normal membranes after incubation of RBC for 24 hr in phosphate- buffered saline containing calcium without added glucose. When compared with normal controls, polyacrylamide gel electrophoresis with sodium dodecyl sulfate (PAGE SDS) of all three preparations showed an increase in membrane binding of globin and protein band 4.5 (60,000 molecular weight). In an attempt to identify band 4.5, 14 enzymes were assayed in the RBC membranes. Of these, catalase and lactate dehydrogenase were increased in membranes from HS RBC and from normal cells exposed to calcium. Only catalase, however, was present in sufficient quantity and had the correct subunit molecular weight on PAGE SDS and calcium- dependent membrane binding to account for an appreciable portion of 4.5. Caralase was further identified with a component of band 4.5 by double immunodiffusion using a specific anti-catalase antibody.

Correction method for a concentration effect in the calculation of molecular weight averages from GPC chromatograms

In the calculation of molecular weight averages by GPC, the traditional method uses the calibration curve obtained at the same concentration as the samples, which results in a large degree of disagreement between molecular weight averages at several concentrations. Because of the concentration dependence of peak elution position in gel permeation chromatography of polymers, correct molecular weight averages cannot be obtained if calibration concentrations are the same as sample concentrations. A computation approach which uses calibration curves at finite and zero concentrations and can correct concentration effects is shown. The concentration used varied from 0.1% up to 0.4%. The elution chromatogram was divided into several parts, and concentration of species at each elution point was obtained from a concentration–peak height calibration curve. Molecular weight at the point was obtained from a molecular weight–elution volume calibration curve corresponding to a concentration of species at the point, and molecular weight averages were calculated by using the usual method. Nearly identical values for molecular weight averages could be obtained at different concentrations, and additional support for this approach is that these values for molecular weight averages were in fair agreement with NBS data.

10] Isolation of human testosterone-estradiol-binding globulin

Publisher Summary The method for the isolation of testosterone-estradiol-binding globulin (TeBG) is not only previously unpublished but also still evolving in laboratory. It is justified in departing from custom by presenting new data in Methods in Enzymology, because previously published methods contain major deficiencies resulting in poor yields of impure and inactive products. As the concentration of TeBG in serum is small, an adequate source of starting material is the initial obstacle in purification. Assuming a molecular weight of about 100,000 and a single binding site per molecule, it can be calculated from the binding capacity that the concentration of TeBG in male serum is about 3.2 rag/liter. If the correct molecular weight is 50,000, the concentration would be 6.4 mg/liter. Cohn fraction IV precipitate, which contains active TeBG, is normally discarded by blood centers and is an ideal source of TeBG. The procedure described in this chapter depends on affinity chromatography for the major purification and the presence of Ca2+ at all stages for stabilization of TeBG activity. The affinity column described in the chapter is novel in which the side chain, azodianiline (ADA), is a bifunetional reagent permitting the facile attachment of ligands and the chemical separation of the ligand from the gel by cleavage of the azo bond with dithionite.

Light scattering as a tool in the study of multicomponent polymer systems

AbstractThe importance of the refractive index increment measured after the establishment of dialysis equilibrium between the polymer solution and the mixed solvent for the evaluation of light scattering measurements from polymer solutions in multicomponent solvents is stressed. The feasibility of determination of heterogeneity in copolymer composition by light scattering can often be considerably extended by using mixed solvents. Light scattering measurements from solutions of binary polymer mixtures yield correct molecular weights of the components and parameters characterizing the thermodynamic interaction of unlike polymer molecules. In all the types of measurements discussed, information is obtained from ratios or differences of rather close values. In order to get physically meaningful data a higher experimental accuracy is required than in conventional light scattering measurements.

Changes in the light-scattering properties of DNA on denaturation

Light scattering measurements made at angles above 25° do not give the correct molecular weight for native DNA. By construction of a low-angle instrument and using new methods to clarify the sample, measurements could be made down to 10°. The low-angle data (10–25°) for calf-thymus and T7 DNA gave a molecular weight twice that obtained at high angles (26–60°). The low-angle molecular weights agreed with those obtained from sedimentation and viscosity measurements. Denaturation of DNA by acid, alkali, or heat caused a halving of the molecular weight, a large decrease in the radius of gyration, and a loss of viscosity. This result was only obtained with low-angle measurements; high-angle measurements showed no decrease in molecular weight on denaturation. On lowering the Na + concentration progressively from 10 −1 to 10 −5 M, there was a linear decrease in the T m and an increase in the intrinsic viscosity. Low-angle light scattering showed that at 10 −3 M salt the molecular weight was one-fourth that observed at 10 −1 or 10 −2 M salt, while at 10 −1 and 10 −2 M salt the solutions did not scatter light at all. When the salt concentration of the latter solutions was raised to 10 −1 M, scattering took place and the molecular weight was that expected for denatured DNA. Identical results were obtained when heat-denatured DNA was studied in low salt suggesting that the properties observed are due to the polyelectrolyte effect in single-stranded DNA and not to abnormal denaturation.

Notes on light scattering from dilute poly(vinyl chloride) solutions in a binary theta‐solvent, tetrahydrofuran/water

AbstractTetrahydrofuran (THF) with 8–9 vol.‐% of water (depending on the polymer tacticity) is at 25°C a Θ‐solvent for poly(vinyl chloride). The refractive index increment (dn/dc)μ (i.e. measured after dialysis) for this system is 0.1085 and 0.112 ml/g for λ0 = 546 and 436 nm, respectively. The light scattering measurements yield correct molecular weights of the polymer. The measurements, however, require special care. Evaporation of THF and polymer aggregation in solvents with even slightly negative second virial coefficients are the major source of error.

Characterization of acid-denatured DNA by low-angle light scattering.

AbstractLow‐angle light scattering results reported previously demonstrated that measurements on high molecular weight native DNA must be made at angles below 30° in order to obtain correct molecular weights. Earlier light‐scattering data obtained on denaturated DNA at angles above 30° showed no change in molecular weight upon denaturation, even though other techniques clearly showed that strand separation occurred. This paper reports low‐angle measurements on solutions of calf thymus and T7 DNA denatured under acidic conditions. The results demonstrate that a halving of molecular weight consistent with strand separation is detected by light scattering only when low‐angle data are used to obtain correct molecular weights for native material. As expected from theoretical considerations, the scattering from denatured DNA is a linear function of sin2(θ/2), where θ is the angle of observation. This result shows that anticipated experimental artifacts have no significant effect on the low‐angle measurements and demonstrates that the curvature in the scattering envelope observed for native DNA below 30° is an inherent property of the native molecule.

Molecular weight of DNA from coliphage T7 by electron microscopy

DNA from the coliphage T7 was isolated by phenol extraction method and prepared for electron microscopy by protein monolayer technique, using two hypophases: 0.1 M ammonium acetate and water. In another experiment, the phages were disrupted by osmotic shock and the released DNA was studied by electron microscopy. The average lengths of the phenol-extracted DNA were found to be 12.0 ± 1.0 μ, and 13.1 ± 1.0 μ for 0.1 M ammonium acetate and water hypophases, respectively. From the osmotic shock preparations, the average molecular length was 12.8 ± 0.9 μ. Assuming B crystalline configuration for DNA on electron microscopic grids, the molecular weight of T7 DNA corresponding to the above experimental lengths were 23.0 ± 1.9, 25.2 ± 1.9, and 24.6 ± 1.7 millions, respectively. Comparison of these values with the molecular weights obtained by means of other techniques indicated that the figure corresponding to the water hypophase or osmotic shock prepparations was more reliable. It was, therefore, concluded that the molecular length of DNA prepared by the osmotic shock method was the same as that of isolated DNA prepared with a water hypophase, and that with the assumed conversion factor of molecular length to weight these methods led to the correct molecular weight.