Purification, characterization, and quantitation of the antigen employed in the immunodiffusion test for diagnosis of equine infectious anemia.

Equine infectious anemia (EIA) antigen extracted from the spleen of horses infected with EIA virus was purified by pH treatment, (NH4)2SO4 fractionation and affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A S20,w of 0.51 was determined and a molecular weight of 7600 was calculated from sedimentation equilibrium analysis. The amino acid composition of the pure antigen indicated the antigen is an acidic protein. Employing radical immunodiffusion (RID) and pure antigen a method for quantitating antigen content of antigen containing preparations was developed.     

Characterization of lecithin:Cholesterol acyltransferase from human plasma: II. Physical properties of the enzyme

The physical properties of purified human plasma lecithin:cholesterol acyltransferase (LCAT) were investigated by techniques including analytical ultracentrifugation, ultraviolet spectroscopy, electrofocusing, and circular dichroism. The partial specific volume of LCAT was determined by sedimentation equilibrium ultracentrifugation experiments in H₂O and D₂O solutions (0.702 ml/g). The M_r was 67,000 by sodium dodecyl sulfate (SDS)-gel electrophoresis and 60,000 by sedimentation equilibrium ultracentrifugation. The discrepancy between the two sets of data presumably arose from the glycoprotein nature of the enzyme. Studies of the ultraviolet spectrum indicated that LCAT contained 6.5% ($\text{w}\text{w}$) tyrosine which corresponds to approximately 18 tyrosine residues/mol of LCAT (polypeptide M_r 45,000). Spectrophotometric titration of the ionizable phenolic side chains indicated that nearly all the tyrosine residues were buried at neutral pH while they became gradually exposed at higher pH. The apparent pK of this transition was about 12.0 contrasted with 9.8, the apparent pK of ionization of the free tyrosyl groups.     

Purification of the protein employed in an immunodiffusion test to diagnose infectious bovine rhinotracheitis.

The antigen used in an immunodiffusion test to diagnose infectious bovine rhinotracheitis has been purified by affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A So20,w of 0.749 was determined and a molecular weight of 8900 was calculated from sedimentation equilibrium analysis. The purified antigen formed precipitin lines of identity with crude diagnostic antigen. Purified antigen remained serologically active in the immunodiffusion test after lyophilization and subsequent reconstitution.     

Characterization of the octamer of histones free in solution

The nucleosome “core protein” isolated from chromatin in high-salt solutions (2 m-NaCl) has been characterized in detail. The preparation described yields material which is stable for prolonged periods at either 4 °C or 37 °C. It has an apparent partial specific volume of 0.767 ml/g and a sedimentation coefficient (S_20,w⁰) of 4.77 (±0.04) S. The molecular weight, determined by sedimentation equilibrium, is 107,500 (±7700), which is compatible with an octameric structure of the form (H2A)₂(H2B)₂(H3)₂(H4)₂, as previously proposed.The sedimentation coefficient and molecular weight are very similar to those of cross-linked core protein, which is known to be octameric.     

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.     

Gene 5 protein of bacteriophage fd: A dimer which interacts Co-operatively with DNA

Isolated gene 5 protein from bacteriophage fd-infected Escherichia coli has been shown by sedimentation equilibrium to exist primarily as a dimer under non-denaturing conditions. The dimer was stable under conditions of high ionic strength, extremes in pH, dilution to 0.075 mg/ml, and increased temperature. Gene 5 protein did not undergo the indefinite self-association observed with gene 32 protein.Three lines of evidence for co-operative binding of gene 5 protein to DNA were developed. First, the interaction between gene 5 protein and phage T4 DNA was examined using a nitrocellulose filter assay. Scatchard plots of the binding data indicated that the interaction was co-operative. Similar results were obtained with gene 32 protein. Second, the co-operative binding of both proteins to DNA was shown by the sensitivity of the protein-DNA interaction to increasing ionic strength at various ratios of protein to DNA. Finally, by using the cross-linking agent, dimethyl suberixmidate, oligomeric structures containing at least seven monomers were found when the DNA was less than saturated.The possibility that gene 5 protein dimers undergo indefinite self-association in the presence of oligonucleotides was examined by sedimentation equilibrium. With oligo[d(pT)₄], the protein dimer was complexed with this oligonucleotide but no self-association was observed. With oligo[d(pT)₈], gene 5 protein formed tetramers, but no significant indefinite association was noted. These results do not suggest a DNA-induced conformational change, which results in indefinite association. A model for the co-operative binding of gene 5 protein to DNA is presented.     

Detergent solubilized and molecular weight estimation of tumor specific surface antigen from SV40 virus transformed cells.

Mild detergent treatment of SV40 transformed mouse embryo fibroblasts, followed by (NH₄)₂SO₄ precipitation of solubilized proteins and rate zonal centrifugation in a sucrose gradient, provided direct and rapid determination of the sedimentation coefficient (4S) of tumor specific surface antigen(s) (TSSA), as detected by an antibody dependent, cell lysis assay. A molecular weight range of 50,000–60,000 was estimated for the TSSA. These results agree with those also obtained from Sephadex G-150 exclusion chromatography.     

A separation chamber to sort cells,nuclei, and chromosomes at moderate g forces. II. Studies on velocity sedimentation and equilibrium density centrifugation of mammalian cells

A separation chamber having a surface of 50 cm² and a height of 2 cm is described for the rapid separation of cells and cell organelles at acceleration forces from 10 to 90g. To eliminate wall sedimentation artifacts, the chamber was positioned 20 cm from the rotor axis in a speed-controlled centrifuge. The chamber has flow deflectors for the undisturbed introduction of the sample layer and the gradient; an antivortex cross prevents swirling upon acceleration and deceleration. To illustrate the use of the separation chamber, examples of velocity sedimentation and of equilibrium density centrifugation are given: (i) human monocytes (70% were 90% pure) are separated from lymphocytes in 10 min at 20g; (ii) nonparenchymal rat liver cells are separated in 10 min at 16g in 97% pure endothelial cells and 99% pure Kupffer cells; (iii) equilibrium density centrifugation of human peripheral blood cells at about 90g permits the separation of erythrocytes, monocytes, lymphocytes, neutrophils, eosinophils, and basophils in one run. B cells are separated from T cells. The movement of swinging buckets is analyzed in mathematical terms and a simple method is offered to determine the position of cells in density gradients with the use of a small programmable calculator.     

Purification and properties of barley leaf ribonuclease

The principal ribonuclease from young barley plants was purified 29 200-fold by a six-step procedure. The enzyme showed a high specific activity (15 5OO ΔA₂₆₀ units/min/mg protein) and a molecular weight of about 25 000 was indicated by gel filtration and equilibrium sedimentation. Kinetic analysis of the cleavage of dinucleoside monophosphates and of yeast RNA indicated a base preference of Gua > Ade ≥ Ura ? Cyt, and was sensitive to the base located on either side of the phosphodiester bond. The enzyme resembles the Type I class of plant ribonucleases (E.C. 2.7.7.x).     

Analysis of 6-Keto PGF1a, 5-hete and LTC4 in rat lung: Comparison of GC/MS,RIA, nad EIA

The recent avaibility of fast and sensitive radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures to measure icosanoids has led to utilization of these techniques by many investigators. A major concern has been that techniques based on immunoreactivity may lack specificity, in particular if complex biologic fluids or tissue extracts are evaluated. The purpose of this investigation was the comparison of icosanoid measurements obtained either with EIA or RIA with those obtained by gas chromatography/mass spectrometry (GC/MS). Rats were injected with Salmonella enteritidis endotoxin, killed at various times after the injection and the ling extract assayed for 6-keto-PGF_1a, 5-HETE and LTC₄.By EIA lung tissue was found to contain large quantities of 6-keto-PGF_1a after endotoxin stimulation. Comparisons made between EIA and GC/MS analysis showed good correlation between 6-keto-PGF_1a amounts in lung as determined by each technique. It was also determined that little purification of lung extract was needed to obtain reliable quantitation of 6-keto-PGF_1a, probably due to the specificity of the antibody and the large quantity of this prostaglandin produced. Crudely purified (Sep-Park) lung extracts gave 5-HETE levels by RIA which were highly correlated with GC/MS values, but RIA values were 70% higher than those obtained by GC/MS. The presence of other components in lung extract which cross react with this 5-HETE antibody was probably responsible for the higher obtained by RIA. LTC₄ was measured by immunoassay in crude lung extracts, as well as after Sep-Pak purification and HPLC purification. LTC₄ levels were identical in unpurified lung extract and after Sep-Pak purifucation, but decreased substantially after HPLC purification. Thus, by validating the icosanoid immunoassays, we have found that they can give accurate and reproductive results in lung tissue, although LTC₄ and 5-HETE must be purified prior to analysis.     

Rat plasma 25-hydroxyvitamin D3 binding protein: An inhibitor of the 25-hydroxyvitamin D3-1 α-hydroxylase

An antibody was prepared from serum of rabbits injected with a pure inhibitor protein obtained from rat serum for chick renal 25-hydroxyvitamin D₃-1α-hydroxylase. The antibody was separated from the endogenous inhibitor in rabbit serum. The antibody shows a single precipitin line with the rat serum antigen and with crude calf serum. Furthermore, the antibody removes the 4.0 S 25-hydroxyvitamin D₃ binding protein from rat serum. The removal of the 25-hydroxyvitamin D₃ binding protein from rat serum with antibody brings about a proportionate removal of inhibitor of the 25-hydroxyvitamin D₃-1α-hydroxylase. The pure inhibitor binds 25-hydroxyvitamin D₃, as demonstrated by sucrose density gradient sedimentation, and shows specificity of binding identical to the serum transport globulin for 25-hydroxyvitamin D₃. Thus, the previously reported inhibitor of the 25-hydroxyvitamin D₃-1α-hydroxylase in rat preparations is the serum 25-hydroxyvitamin D₃ transport protein or some derivative thereof. The antibody added to rat renal mitochondrial preparations does increase the activity of the 1- and 24-hydroxylases slightly but not markedly.     

Ultracentrifugation studies on the dissociation of chemically modified catalases

The dissociation of a series of bovine catalases, in which a proportion of the carboxylic acid groups of glutamic and aspartic acids have been chemically modified by coupling with glycine methyl ester (GME) or ethylenediamine (ED), has been investigated by sedimentation rate and equilibrium methods. Sedimentation equilibrium measurements on GME derivatives have been analysed in terms of a monomer-dimer-trimer- tetramer model. The results show that the association of monomeric (M₁) catalase subunits is consistent with the equilibria 4M₁?2M₂?M₄. The Gibbs energies of association at 284K of the monomeric subunit to dimes (M₂) and tetramers (M₄) were found to be in the range ? 28 to ? 30 kJ mol^?1 and ? 91 to ? 97 kJ mol ^?1, respectively. The Gibbs energy for association of dimer to tetramer is in the range ? 32 to ? 34 kJ mol^?1. Chemical modification of bovine catalase markedly increases its susceptibility to dissociation by sodium n-dodecyl sulphate (SDS) and sedimentation rate measurements suggest that the initial event on addition of SDS is the dissociation of the whole molecule to half-molecules     

Serum and fecal steroid analysis of ovulation,pregnancy, and parturition in the black rhinoceros (Diceros bicornis)

Studies were conducted to determine: (1) if fecal hormone metabolite concentrations correlated with serum estrogen and progesterone concentrations, follicular activity and reproductive behavior in the black rhinoceros (Diceros bicornis) and (2) if threshold values of respective fecal metabolite concentrations correlated with pregnancy. Blood and fecal samples were collected, in conjunction with transrectal ultrasound and behavior observations, for an 18-month period from one black rhinoceros female. Subsequently, serial fecal samples were collected from 13 females in 10 zoos. Quantitative analysis of serum progesterone (P₄) and estradiol (E₂) was performed by radioimmunoassay (RIA): analysis of fecal estrogen metabolites (E) and fecal progesterone metabolites (P) were performed by enzyme immunoassay (EIA). Serum P₂ concentrations identified two luteal phase patterns and two nadirs which corresponded with behavioral estrus. Fecal E patterns indicated a sharp peak which corresponded with breeding. concentrations of fecal P illustrated identifiable nadirs and several peaks which corresponded to serum P₄ nadirs and luteal phases. Serum P₄ concentrations were not different between the luteal phase and pregnancy. Fecal P concentrations started to rise above luteal phase concentrations approximately 150 days postbreeding and remained elevated until immediately before parturition. Serum E₂ and fecal E concentrations rose and subsequently declined after parturition. In the fecal samples from seven pregnant females, fecal P concentrations were similarly elevated compared to six nonpregnant females. Results indicated that fecal steroid metabolites accurately reflected serum steroid hormone concentrations and that the measurement of P and E concentrations permitted the characterization of the estrous cycle, the diagnosis of pregnancy, and the onset of parturition. Zoo Biol 16:121–132, 1997. © 1997 Wiley-Liss, Inc.     

Structural and physicochemical analysis of the reaction between the anti-lysozyme antibody D1.3 and the anti-idiotopic antibodies E225 and E5.2

The reaction between the mouse (BALB/c) anti-idiotiopic monoclonal antibodies E225 and E5.2 and idiotopes on the (BALB/c) anti-lysozyme monoclonal antibody D1.3 has been characterized by titration calorimetry, by equilibrium sedimentation and by the determination of binding association and dissociation rates. The reaction between E5.2 and D1.3 is driven by a large negative enthalpy and its rate and equilibrium association constants are comparable to those observed in other antigen–antibody reactions. In contrast, the reaction between E225 and D1.3 is entropically driven and characterized by slow association kinetic (1 × 10³ M^?1 sec^?1) and a resulting low equilibrium constant (K_a = 2 × 10⁵M ^?1). A correlation of these properties with the three-dimensional structure of the Fab225-FabD1.3 complex, previously determined by X-ray diffraction methods to 2.5 Å resolution, indicates that conformational changes of several D1.3 contacting residues, located in its complementarity determining regions, may explain these features of the reaction.     

Human glucose 6-phosphate dehydrogenase: Purification and characterization of negro type variant (A+) and comparison with normal enzyme (B+)

Human glucose 6-phosphate dehydrogenase (d-glucose 6-phosphate: NADP oxidoreductase, EC 1.1.1.49) (A+), an electrophoretically distinguishable variant found in Negroes, was purified by column chromatographic techniques. The sedimentation patterns of analytical ultracentrifugation and interference patterns of sedimentation equilibrium indicate a homogeneous preparation. The molecular weight (by sedimentation equilibrium method) was estimated as 230,000, which was closely similar to that of the normal wild type enzyme (B+). The sedimentation constant of the variant enzyme (S _20,w=9.0) was smaller than that of the B+ enzyme (S _20,w=10.0). The molecular weight was about 45,000 in 4 mguanidine hydrochloride, indicating that the A+ enzyme, as well as the B+ enzyme, consisted of six subunits of similar size. The optimal pH of the variant enzyme was slightly higher than that of the B+ enzyme. In contrast to the B+ enzyme, magnesium ion increased the A+ enzyme activity with NAD as substrate. The Michaelis constants and the turnover rate were similar to those of the B+ enzyme. The A+ enzyme was serologically indistinguishable from the B+ enzyme when the anti-B+ serum was used as antibody. No significant difference was found in the amino acid composition of acid hydrolysates of the B+ and the A+ enzymes. This does not exclude an amino acid substitution, and, in fact, a single amino acid substitution, i.e., asparagine in B+ and aspartic acid in A+ enzyme, has been found and is being being reported separately.Supported by Research Grant HD-02497-01 and H-3901 from the National Institutes of Health.     

Physical properties of the bovine encephalitogenic protein; molecular weight and conformation

Abstract— The highly basic encephalitogenic protein isolated from bovine spinal cord was studied by various physicochemical methods:

• 1 The molecular weight was determined by sedimentation equilibrium, by calculation from the data on sedimentation coefficients and intrinsic viscosities, by measurement of intrinsic viscosity in the presence of concentrated guanidine hydrochloride (according to the method of Tanford , Kawahara and Lapanie , 1967), and by exclusion chromatography on Sephadex G-100 columns. All values obtained were in good agreement and indicated a molecular weight of approximately 18,000–20,000.

• 2 Studies of sedimentation velocities in the presence and absence of 6 m -guanidine-HCl indicated that there was a significant difference in the values of sedimentation coefficients.

• 3 The same conditions were applied to the measurements of viscosity; the difference was small but significant. These findings and the magnitude of the intrinsic viscosity suggested that this protein was in a disordered configuration. From these data, it is concluded that the protein was apparently monodispersed, in the presence or absence of the denaturing agent. This protein behaved like a polyelectrolyte in neutral aqueous solution.

• 4 The measurements of optical rotatory dispersion also confirmed that this protein existed in a disordered configuration.

     

MOLECULAR WEIGHT AND ELECTROPHORESIS OF CRYSTALLINE RIBONUCLEASE

Electrophoretic studies on purified crystalline ribonuclease showed the absence of any impurities differing in mobility from the bulk of material. The isoelectric point of ribonuclease was found by electrophoresis to be at about pH 7.8. Ultracentrifuge studies indicated fair homogeneity of ribonuclease in solution. Only one moving component has been observed. The molecular weight of ribonuclease was found to be 12,700 from rate of sedimentation (S ²⁵ = 1.85 x 10^–13 in 0.5 M (NH₄)₂SO₄) and diffusion measurement (D = 1.36 x 10_–6 in 0.5 M (NH₄)₂SO₄), in good agreement with the average value of 13,000 found from equilibrium measurements. This low value for the molecular weight of a protein would seem to discredit the value 17,600 as representing a universal unit weight for proteins in general.     

Hydrodynamic properties of monomeric cytochromes P-450LM2 and P-450LM4 in n-octylglucoside solution

Sedimentation equilibrium and sedimentation velocity measurements were carried out on cytochrome P-450_LM2 from phenobarbital-treated rabbit liver and on cytochrome P-450_LM4 from 5,6-benzoflavone-treated rabbit liver in the presence of the nonionic detergent $\text{1-O-}\text{n}\text{-}\text{octyl}\text{-β-}\text{D}\text{-}\text{glucopyranoside}$. P-450_LM2 was monomeric with a molecular weight of 48,800 and a Stokes radius of 3.1 nm in 7 g/l detergent and P-450_LM4 was monomeric with a molecular weight of 49,800 and a Stokes radius of 2.6 nm at 5 g/l detergent. Both particles were spherical in shape under these conditions. Neither cytochrome was irreversibly denatured at these detergent concentrations as indicated by the ability to form substantial amounts (>60%) of the CO adduct with an absorption maximum at 451 nm (P-450_LM2) or 448 nm (P-450_LM4) when diluted into detergent-free buffer containing CO and sodium dithionite.     

Physicochemical studies on xylinan (acetan). III. Hydrodynamic characterization by analytical ultracentrifugation and dynamic light scattering

A laboratory-made sample of the polysaccharide xylinan (acetan) has been further characterized with respect to (i) purity, (ii) molar mass and polydispersity, and (iii) gross conformation by a combination of hydrodynamic measurements (sedimentation velocity and equilibrium analytical ultracentrifugation, viscometry, and dynamic light scattering) in aqueous NaCl (I = 0.10 mol·L⁻¹). Sedimentation velocity diagrams recorded using Schlieren optics revealed highly pure material sedimenting as a single boundary [s^o_20.w = 9.5 ± 0.7) S; k_s = (273 ± 112) mL/g]. The hypersharp nature of these boundaries is symptomatic of a polydisperse and highly nonideal (in the thermodynamic sense) system. Low speed sedimentation equilibrium in the analytical ultracentrifuge using Rayleigh interference optics and two different types of extrapolation procedure (involving point and whole-cell molar masses) gave a weight average molar mass M_w of (2.5 ± 0.5) × 10⁻⁶ g·mol⁻¹ and also a second virial coefficient, B = (2.8 ± 0.7) × 10⁻⁴ mL·mol·g⁻², both values in good agreement with those from light scattering-based procedures (Part II of this series). A dynamic Zimm plot from dynamic light scattering measurements gave a z-average translational diffusion coefficient D^o_20.w = (3.02 ± 0.05) × 10⁻⁸ cm²·s⁻¹ and the concentration-dependence parameter k_D = (370 ± 15) mL/g. Combination of s^o_20.w with D^o_20.w via the Svedberg equation gave another estimate for M_w of ≅ 2.4 × 10⁶ g/mol, again in good agreement. Both the Wales-van Holde ratio (k_s/[η]) ≅ 0.4 (with [η] = (760 ± 77) mL/g) and the ρ-parameter (ratio of the radius of gyration from static light scattering to the hydrodynamic radius from dynamic light scattering) as ρ > 2.0 all indicate an extended conformation for the macromolecules in solution. These findings, plus Rinde-type simulations of the sedimentation equilibrium data are all consistent with the interpretation in terms of a unimodal wormlike coil model performed earlier. © 1996 John Wiley & Sons, Inc.     

Purification,Crystallization and Some Properties of Intracellular Pullulanase from Aerobacter aerogenes

Intracellular pullulanase was entirely extracted with sodium dodecylsulfate from the cells and was purified by means of ammonium sulfate fractionation and DEAE-cellulose and Sephadex chromatography. Crystalline pullulanase was precipitated with saturated ammonium sulfate solution. Intracellular pullulanase was purified over 150 fold in 17% yield to a final specific activity of 7000 per mg protein from the enzyme solution obtained by SDS-extraction. On ultracentrifugation analysis, the enzyme showed a symmetrical peak. The sedimentation coefficient, s_{20, w} was 6.29 S. Polyacrylamide disc electrophoresis gave a main band and a sub-band, and both showed activity. Molecular weight of intracellular pullulanase was estimated to be (8±1) × 10,000 from gel filtration with Sephadex G-200 and to be (9±1) × 10,000 from sedimentation equilibrium. These values were higher than that (6~7 × 10,000) of extracellular pullulanase. Both enzymes differed slightly in thermal- and pH-stabilities.